Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi.

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.

In vitro differentiation of Trypanosoma cruzi under chemically defined conditions.

Metacyclic trypomastigotes of Trypanosoma cruzi have been obtained in chemically defined axenic culture. The differentiating medium, composed of artificial triatomine urine supplemented with proline, allows high yields of metacyclic trypomastigotes after 72-h incubation of T. cruzi cells at 27 degrees C. Morphological differentiation of the parasites is gradual under these chemically defined conditions and is preceded by the expression of stage-specific polypeptides. The yield of in vitro-induced metacyclic trypomastigotes depends upon the age of the epimastigote culture, the size of the inoculum and the depth of the medium. Metacyclic trypomastigotes differentiated in vitro from the Dm 28c clone of T. cruzi are both resistant to complement lysis and to macrophage digestion. They are able to infect mice with an efficiency similar to that obtained for natural metacyclic trypomastigotes obtained from triatomine excreta.

Reiteration frequency of procollagen genes in the guinea pig genome. Collagen genes are not amplified during granuloma fibroblasts differentiation.

Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen 125I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot 1/2 800-900 mol . s . 1-1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11-13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.

Use of acrylic of low polymerization after heminandibulectomy.

Two cases of mandibular reconstruction are presented in which a premolded prosthesis of acrylic of low polymerization covered by silicone rubber was used. We believe that the good results obtained were not only due to the material used, but also because the periosteum and meniscus were preserved in the joint space.

Does early oral feeding increase the likelihood of salivary fistula after total laryngectomy?

Objective: This study compared the incidence of salivary fistula between groups with an early or late reintroduction of oral feeding, and identified the predictive factors for salivary fistula. Methods: A randomised trial was performed using 89 patients with larynx or hypopharynx cancer, assigned to 2 groups (early or late). In the early group, oral feeding was started 24 hours after total laryngectomy or total pharyngolaryngectomy, and in the late group, it was started from post-operative day 7 onwards. The occurrence of salivary fistula was evaluated in relation to the following variables: early or late oral feeding, nutritional status, cancer stage, surgery performed, and type of neck dissection. Results: The incidence of salivary fistula was 27.3 per cent (n = 12) in the early group and 13.3 per cent (n = 6) in the late group (p = 0.10). The following variables were not statistically significant: nutritional status (p = 0.45); tumour location (p = 0.37); type of surgery (p = 0.91) and type of neck dissection (p = 0.62). A significant difference (p = 0.02) between the free margins and invasive carcinoma was observed. Conclusion: The early reintroduction of oral feeding in total laryngectomised patients did not increase the incidence of salivary fistula.

Prognostic factors for surgically resected squamous cell carcinoma of the lower oral cavity.

AIM: The objective of this study was to evaluate the impact of clinical variables on survival rates in patients with squamous cell carcinomas in the lower oral cavity. METHODS: We conducted a retrospective study of patients who exhibited a histological diagnosis of squamous cell carcinoma of the lower oral cavity, who underwent surgery. Data on clinical, histological, and treatment variables were collected to assess the effect of these variables on global survival and disease-free curves. RESULTS: We studied 117 patients, 86 (73.5%) males and 31 (26.5%) females, who underwent surgery between January 2005 and June 2009. The overall survival rate was 74.4% after two years, and the disease-free rate was 67.5%. Midline invasion by the primary tumor negatively impacted overall survival (P=0.02) and disease-free survival (P=0.01). The disease-free survival rate of patients with histologically confirmed neck metastases was 76.8% versus 59% for patients without metastases (P=0.01). Disease-free survival of patients with one affected lymph node was 89.5%, and 45.2% for more than one affected lymph node (P<0.01). Patients who ended radiotherapy in less than 60 days exhibited a disease-free rate of 59.0%, and patients whose treatment lasted more than 60 days exhibited a rate of 77.1% (P=0.06). CONCLUSION: In our study, primary tumor invasion of the midline, the presence of more than one affected lymph node as confirmed by histology, the delaying radiotherapy for more than 56 days and radiotherapy that lasted longer than 60 days worsen patient prognosis.

Further studies on collagen mRNA: partial chemical characterization and polyadenylic acid sequence.

Collagen mRNA has already been purified and characterized by us. Its purity has now been enhanced by two different methods. Gel electrophoresis shows in either method, a single peak with the same mobility already reported: 1.05 X 10(6) daltons. Base composition analyses of collagen mRNA purified by either method were almost identical. Chemical analyses of the isolated polyadenylic acid stretch show that it is, 0.48 X 10(5) daltons-long, (about 140 nucleotides-long), contains 75% AMP, and is located at the 3' end of the polymer.

Characterization and expression of proteases during Trypanosoma cruzi metacyclogenesis.

Investigation of protease activities during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigoes (metacyclo-genesis) revealed three major components with apparent molecular weights of 65, 52, and 40 kDa. The 65-kDa protease is a metacyclic trypomastigote stage-specific protease with an isoelectric point of 5.2 whose activity is inhibited by 1,10-phenanthroline, suggesting that it might be a metalloprotease. The 52-kDa component is also a metalloprotease which is constitutively expressed in epimastigotes and metacyclic trypomastigoes. On the other hand, the 40-kDa component is apparently made up of several isoforms of a cysteine protease which is expressed in much higher levels in epimastigotes than in metacyclic trypomastigote forms. The fact that the 65- and 40-kDa proteases are developmentally regulated suggests that proteases might be important for T. cruzi differentiation. Accordingly, T. cruzi metacyclogenesis is blocked by metallo- and cysteine-protease inhibitors.

Haemolymph and fat body metallo-protease associated with Enterobacter cloacae infection in the bloodsucking insect, Rhodnius prolixus.

Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.

Expression and polymorphism of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen.

The study of the expression of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen (CRA) during the metacyclogenesis process shows that this gene is not expressed in metacyclic trypomastigote forms of the parasite. However, a slight increase in CRA expression was observed following the nutritional stress of epimastigotes which precedes T. cruzi metacyclogenesis in vitro. The comparison of the expression of CRA in different T. cruzi strains shows that this gene is highly polymorphic: some strains display one and others display two polypeptides reacting with a CRA antiserum. The comparison of T. cruzi G-49 strain and Dm 28c clone shows that they display rather different Northern and Southern blot profiles when probed with a clone corresponding to the repetitive region of the CRA gene. A similar polymorphism was also observed for the gene encoding a flagellar repetitive antigen, suggesting that gene polymorphism might be a common feature of many T. cruzi genes.

Isolation and characterization of collagen messenger RNA*.

Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns.

Haemolymph and fat body metallo-protease associated with Enterobacter cloacae infection in the bloodsucking insect, Rhodnius prolixus

Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.