Leonardo Rodrigues

Diversity and frequency of yeasts from the dorsum of the tongue and necrotic root canals associated with primary apical periodontitis

AIMS To determine the frequency and diversity of yeasts from the dorsum of the tongue and necrotic root canals with teeth associated with primary apical periodontitis. METHODOLOGY Detailed medical and dental histories of 168 patients were recorded. The samples were collected from the dorsum of tongue and from 168 teeth with root canals contained necrotic pulps. Yeasts were isolated on Sabouraud agar with 100 mg L(-1) chloramphenicol, purified and characterized by standard methods. Identification was confirmed by EI1 PCR fingerprint technique. Yeast isolates of uncertain identity or with a different genetic fingerprint profile from the reference strains were identified by sequencing the D1/D2 variable domains of the large subunit rDNA. RESULTS Yeasts were isolated from 22.6% of teeth sampled and from 45.8% of tongue samples. Candida albicans was the most frequently isolated species at both investigated sites but other species were also found. Saccharomyces cerevisiae and Kluyveromyces lactis were recovered from the tongue. CONCLUSIONS Although the detection of yeasts in the root canal does not imply an involvement in the disease process, the study suggests a frequency of Candida spp. in primary endodontic infections that deserves further clarification.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis.

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Participation of N-acetyl-D-glucosamine carbohydrate moieties in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria tenagophila

Lectin-carbohydrate binding may be involved in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria; therefore, we tested if this interaction is associated with snail resistance against Schistosoma infection. In vitro data showed that most of the S. mansoni sporocysts cultured with haemocytes from Biomphalaria glabrata BH, a highly susceptible snail strain, had a low number of cells that adhered to their tegument and a low mortality rate. Moreover, the addition of N-acetyl-D-glucosamine (GlcNAc) did not alter this pattern of adherence and mortality. Using haemocytes and haemolymph of Biomphalaria tenagophila Cabo Frio, we observed a high percentage of sporocysts with adherent cells, but complete encapsulation was not detected. Low concentrations of GlcNAc increased haemocyte binding to the sporocysts and mortality, which returned to basal levels with high concentrations of the carbohydrate. In contrast, haemocytes plus haemolymph from B. tenagophila Taim encapsulated cellular adhesion index of level 3 and destroyed over 30% of the S. mansoni sporocysts in culture. Interestingly, the addition of GlcNAc, but not mannose, to the culture medium resulted in the significant inhibition of cellular adhesion to the parasite tegument and the reduction of parasite mortality, suggesting that GlcNAc carbohydrate moieties are important to the recognition of S. mansoni by B. tenagophila Taim.

Distribution of the endophytic fungi community in leaves of Bauhinia brevipes (Fabaceae)

Endophytic fungi represent large, yet unexplored components of biodiversity. This work evaluated the richness and the distribution of endophytes in the leaves of Bauhinia brevipes (Fabaceae). A total of 1110 colonies were recovered from the samples and grouped by their morphological traits into 126 taxa. The total number of taxa according to leaf development was: 102 in mature leaves, 93 in recently expanded leaves and 79 for unfolded leaves. The major endophyte genera were Phomopsis, followed by Dothiorella, Pestalotiopsis and Acremonium. The richness and the isolate numbers of endophytes were not statistically affected by leaf region. However, some taxa were leaf-age specific; six were isolated only from unfolded leaves, nine from recently expanded leaves and 17 were exclusively found in mature leaves. The composition of endophytes varied with leaf region; the similarities (Jaccards Index) among the leaf regions of different leaf ages ranged from 0.36 to 0.46, indicating a high spatial variation in the community of endophytic fungi inside the leaves. The high richness of endophytes in this host plant highlights a significant contribution of fungi to tropical biodiversity and the need for further research in this area.

Synthesis, characterization and biocidal activity of new organotin complexes of 2-(3-oxocyclohex-1-enyl)benzoic acid.

The reaction of 1,3-cyclohexadione with 2-aminobenzoic acid has produced the 2-(3-oxocyclohex-1-enyl)benzoic acid (HOBz). Subsequent reactions of the ligand with organotin chlorides led to [Me(2)Sn(OBz)O](2) (1), [Bu(2)Sn(OBz)O](2) (2), [Ph(2)Sn(OBz)O](2) (3), [Me(3)Sn(OBz)] (4), [Bu(3)Sn(OBz)] (5) and [Ph(3)Sn(OBz)] (6). All complexes have been fully characterized. In addition the structure of complexes (2) and (4) have been authenticated by X-ray crystallography. The biological activity of all derivatives has been screened against Cryptococcus neoformans and Candida albicans. In addition we have performed toxicological testes employing human kidney cell. The complexes (3), (5) and (6) displayed the best values of inhibition of the fungus growing, superior to ketoconazole. Compound (5) presented promising results in view of the antifungal and cytotoxicity assays.

Differential Proteinase Patterns among Candida albicans Strains Isolated from Root Canal and Lingual Dorsum: Possible Roles in Periapical Disease

INTRODUCTION Proteinases play pivotal roles in Candida albicans infections. Although the yeast can colonize the pulpal environment, there is no information about the enzymatic profile of this organism. This in vitro study aimed to determine the proteolysis levels and to investigate differences in the expression of aspartyl proteinase genes (Sap 1, Sap 2, and Sap 4) among various root canal strains and clinical isolates from the lingual dorsum. METHODS The extracellular proteinase activity of 104 C. albicans samples isolated from the lingual dorsum and from necrotic root canals was measured with respect to bovine serum albumin degradation after 5 days of incubation at 37°C. We used reverse-transcription polymerase chain reaction, a highly sensitive method, to detect messenger RNA transcripts of aspartyl proteinase genes (Sap 1, Sap 2, and Sap 4). The C. albicans strain SC 5314 was used as a positive control for both experiments because it is recognized as being highly proteolytic. All tests were performed in triplicate. RESULTS Regardless of the isolation site, all C. albicans strains produced an opaque precipitation halo around the colonies, indicating some proteinase activity. However, the production of proteinase on the plates was significantly greater (P < .05) by the endodontic samples. Sap 2 was the most commonly expressed gene in all samples. Among the root canal samples, the detection of Sap 1 transcripts was always associated with the expression of Sap 2 and Sap 4. Sap 4 gene expression was detected in all root canal samples. The simultaneous expression of the 3 investigated Sap genes (Sap 1, Sap 2, and Sap 4) was more common in strains isolated from the lingual dorsum (50%) than in those isolated from root canals (29.4%). CONCLUSIONS The increased proteolytic activity as well as the distinct pattern of Sap expression observed among the root canal samples may suggest a pathogenic role for C. albicans in endodontic infections.

BRAF V600E and loss of heterozygosity assessment in benign oralneural tumours

BACKGROUND The purpose of this study was to evaluate loss of heterozygosity (LOH) and to assess BRAF V600E mutation in oral neurofibromas, palisaded encapsulated neuromas (PEN) and schwannomas. METHODS Six oral neurofibroma, 5 PEN and 3 schwannoma samples were included in the study. LOH was assessed using polymorphic microsatellite markers at chromosome regions 3p (marker D3S1029), 9p (markers D9S171, D9S162, D9S157), 11q (marker D11S1369), and 17p (markers AFM238WF2 and P53), and results were evaluated after capillary electrophoresis. BRAF mutation encoding V600E was assessed by real-time PCR with a specific TaqMan probe to detect the T>A transversion at position c.1799. RESULTS LOH occurred at chromosomes 3p (marker D3S1029), 11q (D11S1369) and 17p (AFM238WF2 and P53). LOH occurred in 2/6 neurofibromas, 2/5 PEN and in none of the 3 schwannoma samples. The 6 neurofibromas, 2/2 PEN evaluated and the 3 schwannomas were BRAF wild type. CONCLUSION According to our results, oral benign peripheral nerve sheath tumours have a low LOH rate, but P53 locus alteration is occasionally found. Additionally, BRAF V600E mutation is either not relevant to the molecular pathogenesis of this group of lesions of the oral cavity, or may occur at very low rates.