Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.     

Histology of the kidney and urinary bladder of Siphonops annulatus (Amphibia-Gymnophiona)

The histology of the kidney and urinary bladder of Siphonops annulatus was studied by light microscopy in semithin sections of tissue embedded in hydrophilic resin. The kidney's nephron comprises the renal corpuscle, neck segment, proximal tubule, intermediate segment, distal tubule and collecting tubule. Nephrostomes are present. This structure, the neck segment, and intermediate tubules present long cilia, and probably play important roles in the propulsion of the peritoneal fluid and glomerular filtrate. The proximal tubule cells possess loosely packed microvilli and contain abundant polymorphic granules and vesicles that assume the aspect of lysosomes in different stages of intracellular digestion. The distal tubules are characterized by large, vertically disposed mitochondria assuming the aspect of ions transporting cells. The urinary bladder is lined with a transitional epithelium, whose aspect varies according to the quantity of urine.     

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization

The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.     

Molecular epidemiologic evaluation of endocarditis due to Oerskovia turbata and CDC group A-3 associated with contaminated homograft valves

Oerskovia turbata is an unusual bacterial cause of endocarditis and septicemia in immunocompromised patients. In this study, we compared 12 isolates from a 1975 medical center cluster, 11 originally identified as O. turbata (four from the blood of a homograft aortic valve-associated endocarditis patient and seven from contaminated homograft valves) and one CDC group A-3 strain from the blood of a second endocarditis patient with fatal outcome, with eight control strains from unrelated locations. The control strains included type and reference strains of O. turbata, Cellulomonas hominis, and CDC group A-3. The four blood isolates from the first patient and six of the valve isolates shared identical biochemical, antimicrobial susceptibility, and BglI ribotype patterns that differed from the second patient's isolate and control strains. The blood isolate from the second patient and the remaining valve isolate shared a phenotypic and genotypic profile and were phenotypically identical to, but epidemiologically different from, the CDC group A-3 reference strain with the strain-specific enzyme. Also, these isolates differed from the type strain and the other reference strains of C. hominis and O. turbata. Our results indicate that the four blood isolates from the first patient and six of the homograft valve isolates represent a single clone of O. turbata associated with endocarditis. Additionally, our results indicate that the blood isolate from the second patient and one of the homograft valve isolates differ from O. turbata and C. hominis and represent a unique clone of CDC group A-3 associated with fatal endocarditis.     

Microhardness of superficial and deep sound human dentin

Our purpose in this study was to determine the microhardness of superficial and deep dentin by means of two indentation methods (Knoop and Vickers) under two different applied loads. Twelve dentin discs approximately 2-mm thick were obtained from both superficial and deep dentin by transversally sectioning the crowns of sound, extracted human third molars with a diamond blade under water irrigation. Dentin surfaces were sequentially polished, and indentations (n = 20 per surface) were performed with either Vickers indentor at loads of 300 and 500 g, respectively, or Knoop indentor at loads of 50 and 100 g, respectively. Average Vickers hardness number (VHN) and Knoop hardness number (KHN) were calculated and treated with two-way analysis of variance (ANOVA) and Student's t test. Microhardness of dentin was not influenced by the different loads applied for both indentation methods. Knoop hardness was significantly higher for superficial than for deep dentin (p < 0.05). Conversely, Vickers hardness was not significantly different for both substrates (p > 0.05). Differences in dentin hardness as a function of depth exist, but they might not be relevant, and no alteration of the distribution of stresses along the adhesive interface is expected.     

FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified 17 new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that fibroblast growth factor (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs.     

Hepatic gluconeogenesis and Krebs cycle fluxes in a CCl4 model of acute liver failure

Acute liver failure was induced in rats by CCl4 administration and its effects on the hepatic Krebs cycle and gluconeogenic fluxes were evaluated in situ by 13C NMR isotopomer analysis of hepatic glucose following infusion of [U-13C]propionate. In fed animals, CCl4 injury caused a significant increase in relative gluconeogenic flux from 0.80+/-0.10 to 1.34 +/-0.24 times the flux through citrate synthase (p<0.01). In 24-h fasted animals, CCl4-injury also significantly increased relative gluconeogenic flux from 1.36+/-0.16 to 1.80+/-0.22 times the flux through citrate synthase (p<0.01). Recycling of PEP via pyruvate and oxaloacetate was extensive under all conditions and was not significantly altered by CCl4 injury. CCl4 injury significantly reduced hepatic glucose output by 26% (42.8+/-7.3 vs 58.1+/-2.4 micromol/kg/min, p=0.005), which was attributed to a 26% decrease in absolute gluconeogenic flux from PEP (85.6+/-14.6 vs 116+/-4.8 micromol/kg/min, p<0.01). These changes were accompanied by a 47% reduction in absolute citrate synthase flux (90.6+/-8.0 to 47.6+/-8.0 micromol/kg/min, p<0.005), indicating that oxidative Krebs cycle flux was more susceptible to CCl4 injury. The reduction in absolute fluxes indicate a significant loss of hepatic metabolic capacity, while the significant increases in relative gluconeogenic fluxes suggest a reorganization of metabolic activity towards preserving hepatic glucose output.     

Distribution of cytoskeletal structures and organelles of the host cell during evolution of the intracellular parasitism by Trypanosoma cruzi

The distribution of microtubules, microfilaments, mitochondria, Golgi complex and endosomes/lysosomes was analyzed in Vero cells allowed to interact for different periods of time with the pathogenic protozoan Trypanosoma cruzi and observed by confocal laser scanning microscopy. Microtubules were revealed using a mouse monoclonal anti-alpha-tubulin antibody. Actin filaments were revealed using phalloidin-rhodamine. To identify mitochondria, endosomes/lysosomes and the Golgi complex the cells were labelled with Rhodamine 123, Lucifer yellow and C6-NBD-ceramide, respectively. During cell invasion actin filaments concentrate at the site of parasite penetration in some, but not in all cells, probably depending upon the mechanism used by the trypomastigote form to penetrate into the host cells. Following internalization the trypomastigote form gradually changes into the amastigote form, disruption of the parasitophorous vacuole membrane takes place and the amastigote form enters in direct contact with host cell structures and organelles, and starts to divide. The presence of the parasite in the cytoplasm of the host cell did not induce significant changes in the distribution of actin filaments, microtubules, the Golgi complex, mitochondria and endosomes/lysosomes during the first 48 h of infection. Amastigote forms were seen close to the microtubules. After 72 h of interaction, the number of microtubules and microfilaments around the parasites was reduced and lysosomes and mitochondria were seen in between the parasites.