Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization

We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1) binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0º C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse per cent Gull, 1988) indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype).

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism

To further investigate phylogeny of kinetoplastid protozoa, the sequences of small subunit (18S) ribosomal RNA of nine bodonid isolates and ten isolates of insect trypanosomatids have been determined. The root of the kinetoplastid tree was attached to the branch of Bodo designis and/or Cruzella marina. The suborder Trypanosomatina appeared as a monophyletic group, while the suborder Bodonina was paraphyletic. Among bodonid lineages, parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism and supporting the `vertebrate-first hypothesis'. The tree indicated that the genera Cryptobia and Bodo are artificial taxa. Separation of fish cryptobias and Trypanoplasma borreli as different genera was not supported. In trypanosomatids, the genera Leptomonas and Blastocrithidia were polyphyletic, similar to the genera Herpetomonas and Crithidia and in contrast to the monophyletic genera Trypanosoma and Phytomonas. This analysis has shown that the morphological classification of kinetoplastids does not in general reflect their genetic affinities and needs a revision.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Ancient medical texts, modern reading problems

The word tradition has a very specific meaning in linguistics: the passing down of a text, which may have been completed or corrected by different copyists at different times, when the concept of authorship was not the same as it is today. When reading an ancient text the word tradition must be in the reader's mind. To discuss one of the problems an ancient text poses to its modern readers, this work deals with one of the first printed medical texts in Portuguese, the Regimento proueytoso contra ha pestenença, and draws a parallel between it and two related texts, A moche profitable treatise against the pestilence, and the Recopilaçam das cousas que conuem guardar se no modo de preseruar à Cidade de Lixboa E os sãos, & curar os que esteuerem enfermos de Peste. The problems which arise out of the textual structure of those books show how difficult is to establish a tradition of another type, the medical tradition. The linguistic study of the innumerable medieval plague treatises may throw light on the continuities and on the disruptions of the so-called hippocratic-galenical medical tradition.

Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

Knowledge and attitudes of nurses towards alcohol and related problems: the impact of an educational intervention

An exploratory study of quasi-experimental approach that aimed to verify the impact of an educational intervention on attitudes and knowledge of nurses towards alcohol use and associated problems. The sample included 185 nurses, divided into two groups: 84 submitted to a training course and formed the experimental group. Data were collected through a knowledge survey and an attitude scale. The attitudes of the participants of both groups were positive. There were no significant differences between groups in relation to knowledge. The strongest predictors of positive attitudes were possessed preparation to act with chemical dependents (OR = 2.18), "have received increased workload during graduation on the theme, 'alcohol and other drugs'" (OR = 1.70), and "completed graduate school" (OR = 2.59). The educational intervention had a positive impact on the attitudes of nurses towards alcoholics, work and interpersonal relationships with such clientele.

Health problems of nursing workers in a public educational institution

Objective: To identify the issues occurred with nursing workers through a Health Monitoring System for Nursing Workers (SIMOSTE) and to describe the consequences of those problems. Method: This is a quantitative, exploratory and descriptive study realized in a teaching hospital in the west region of the city of São Paulo. Results: From the SIMOSTE, 1.847 occurrences were registered in a six month period. Within the main occurrences, medical licenses, work related accidents with and without removals; psychiatric consultations and psychotherapy were highlighted. Conclusion: The data points out to the need for the development of new health vigilance actions to notify accidents and illness related to work, besides the prevention of issues. 


Papaya pulp gelling: is it premature ripening or problems of water accumulation in the apoplast?

Gelled aspect in papaya fruit is typically confused with premature ripening. This research reports the characterization of this physiological disorder in the pulp of papaya fruit by measuring electrolyte leakage, Pi content, lipid peroxidation, pulp firmness, mineral contents (Ca, Mg and K - in pulp and seed tissues), and histological analysis of pulp tissue. The results showed that the gelled aspect of the papaya fruit pulp is not associated with tissue premature ripening. Data indicate a reduction of the vacuole water intake as the principal cause of the loss of cellular turgor; while the waterlogged aspect of the tissue may be due to water accumulation in the apoplast.

A new method for intraoperative localization of epilepsy focus by means of a gamma probe

Objective To evaluate the utility of a new multimodal image-guided intervention technique to detect epileptogenic areas with a gamma probe as compared with intraoperative electrocorticography. Materials and Methods Two symptomatic patients with refractory epilepsy underwent magnetic resonance imaging, videoelectroencephalography, brain SPECT scan, neuropsychological evaluation and were submitted to gamma probe-assisted surgery. Results In patient 1, maximum radioactive count was initially observed on the temporal gyrus at about 3.5 cm posteriorly to the tip of the left temporal lobe. After corticotomy, the gamma probe indicated maximum count at the head of the hippocampus, in agreement with the findings of intraoperative electrocorticography. In patient 2, maximum count was observed in the occipital region at the transition between the temporal and parietal lobes (right hemisphere). During the surgery, the area of epileptogenic activity mapped at electrocorticography was also delimited, demarcated, and compared with the gamma probe findings. After lesionectomy, new radioactive counts were performed both in the patients and on the surgical specimens (ex-vivo). Conclusion The comparison between intraoperative electrocorticography and gamma probe-assisted surgery showed similarity of both methods. The advantages of gamma probe include: noninvasiveness, low cost and capacity to demonstrate decrease in the radioactive activity at the site of excision after lesionectomy.