Cell proliferation and interferon-gamma response to recombinant MBP-3, NarL, MT-10.3, and 16 kDa Mycobacterium tuberculosis antigens in Brazilian tuberculosis patients

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-gamma (IFN-gamma)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-gamma human T cell immune responses, to four recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%) and controls (> 64%) respond to the PPD, 16 kDa was more specifically recognized (> 21%) although less sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average level of IFN-gamma was induced by 16 kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8 pg/ml x 221.6 pg/ml, respectively), although the difference was not significant. These data show that in contrast with the other recombinant proteins, the stimulatory potency of 16 kDa to induce proliferative and INF-gamma response was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a more selective immune response than PPD.     

Comparison of artificial insemination versus embryo transfer in lactating dairy cows

Conception rates (CR) are low in dairy cows and previous research suggests that this could be due to impaired early embryonic development. Therefore, we hypothesized that CR could be improved by embryo transfer (ET) compared with AI. During 365 days, 550 potential breedings were used from 243 lactating Holstein cows (average milk production, 35 kg/day). Cows had their ovulation synchronized (GnRH-7d-PGF(2alpha)-3d-GnRH) and they were randomly assigned for AI immediately after the second GnRH injection (Day 0) or for transfer of one embryo 7 days later. Circulating progesterone concentrations and follicular and luteal size were determined on Days 0 and 7. Pregnancy diagnosis was performed on Days 25 or 32 and pregnant cows were reevaluated on Days 60-66. Single-ovulating cows with synchronized ovarian status had similar CR on Days 25-32 with ET (n = 176; 40.3%) and AI (n = 160; 35.6%). Pregnancy loss between Days 25-32 and 60-66 also did not differ (P = 0.38) between ET (26.2%) and AI (18.6%). When single (n = 334) and multiple (n = 57) ovulators were compared, independent of treatment, multiple ovulators had greater (P < 0.001) circulating progesterone concentrations on Day 7 (2.7 ng/ml versus 1.9 ng/ml) and there was a tendency (P = 0.10) for a greater CR in multiple ovulators (50.9% versus 38.1%). However, there was no difference in CR between AI and ET cows with multiple ovulations (50.0% versus 51.7%). In single-ovulating cows, CR tended to be lower for AI than ET in cows ovulating smaller follicles (diameter < or = 15 mm; 23.7% versus 42.3%; P = 0.06) but not average-diameter follicles (16-19 mm; 41.2% versus 37.3%; P = 0.81) or larger (> or =20 mm; 34.3 versus 51.0%; P = 0.36) follicles. Thus, although ET did not improve overall CR in lactating cows, follicle diameter and number of ovulating follicles may determine success with these procedures.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

Influence of Hydrological Pulse on Bacterial Growth and DOC Uptake in a Clear-Water Amazonian Lake

This study was conducted to evaluate: (1) the bacterial growth and the dissolved organic carbon (DOC) uptake in an Amazonian lake (Lake Batata) at high-water and low-water periods of the flood pulse; (2) the influence of nitrogen and phosphorus (NP) additions on bacterial growth and DOC uptake in Lake Batata at two flood pulse periods; and (3) the bioavailability of the main DOC sources in Lake Batata. Lake Batata is a typical clear-water Amazonian lake, located in the watershed of Trombetas River, Central Amazon, Brazil. Bacterial batch cultures were set up with 90% 0.2-μm filtered water and 10% inoculum from Lake Batata. N-NH₄NO₃ and P-KH₂PO₄, with final concentrations of 50 and 5 μM, respectively, were added to the cultures, except for controls. Extra sources of DOC (e.g., algal lysate, plant leachates) were added to constitute six distinct treatments. Bacterial response was measured by maximum bacterial abundance and rates of bacterial production, respiration, DOC uptake, and bacterial growth efficiency (BGE). Bacterial growth and DOC uptake were higher in NP treatments than in controls, indicating a consistent nutrient limitation in Lake Batata. The composition of DOC also seems to be an important regulating factor of bacterial growth in Lake Batata. Seasonally, bacterial growth and DOC bioavailability were higher at low-water period, when the phytoplankton is a significant extra source of DOC, than at high-water period, when the forest is the main source of DOC. DOC bioavailability was better estimated based on the diversity and the diagenetic stage of carbon compounds than on single classes of labile compounds. Changes in BGE were better related to CNP stoichiometry in the water, and the “excess” of organic substrates was oxidized in catabolism, despite the quality of these compounds for bacterial growth. Finally, we conclude that bacterial growth and DOC uptake vary throughout the flood pulse in clear-water Amazonian ecosystems as a result of changes in nutrient concentration and in DOC composition.     

Macroinvertebrate succession during leaf litter breakdown in a perennial karstic river in Western Brazil

Leaf litter is a major basal resource to stream ecosystems, but few studies addressed their role in karst systems, mainly in intermittent springs and lakes. Patterns of resource use in perennial rivers are poorly known, although the input of leaf litter strongly influences macroinvertebrate assemblage structure. In this study, we evaluated the structure of macroinvertebrate assemblages along the decomposition of leaf litter in a tropical karst river, using leaf litter cages made of coarse nylon mesh (25 mm) to allow colonization by macroinvertebrates. The experiment was followed weekly for 10 weeks. The assemblages were dominated by snails (90.5% of total fauna), hyalellid amphipods, and larval chironomid midges, with highest abundances in the intermediate stages of the experiment, resulting in a gradient in assemblage structure. The large abundance of snails, which are common in other karst systems, suggest that this group may have an important role in decomposer food webs, facilitating or directly contributing to leaf breakdown. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

A propolis extract and the labeling of blood constituents with technetium-99m

Since ancient times propolis has been employed for many human purposes because to their favourable properties. Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures. Some authors have reported that synthetic or natural drugs can interfere with the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the action of a propolis extract on the labeling of blood elements with 99mTc. Samples of whole blood of male Wistar rats were incubated in sequence with an aqueous propolis extract at different concentrations, stannous chloride and 99mTc, as sodium pertechnetate. Blood samples were centrifuged to separate plasma and blood cells, soluble and insoluble fractions of plasma and blood cells were also separated after precipitation in trichloroacetic acid solution and centrifugation. The radioactivity was counted and the percentage of incorporated radioactivity (%ATI) for each fraction was calculated. The data obtained showed that the aqueous propolis extract used decreased significantly the %ATI in plasma proteins at higher concentration studied. Results suggest that at high concentration the constituents of this extract could alter the labeling of plasma proteins competing with same binding sites of the 99mTc on the plasma proteins or acting as antioxidant compounds.     

The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Trypanosoma rangeli: Differential expression of cell surface polypeptides and ecto-phosphatase activity in short and long epimastigote forms

Trypanosoma rangeli is a parasite of a numerous wild and domestic animals, presenting wide geographical distribution and high immunological cross-reactivity with Trypanosoma cruzi, which may lead to misdiagnosis. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. Here, we characterized the cell surface polypeptides and the phosphatase activities in short and long epimastigotes forms of T. rangeli, using intact living parasites. The surface protein profile revealed by the incubation of parasites with biotin showed a preferential expression of the 97, 70, 50, 45, 25-22, and 15 kDa biotinylated polypeptides in the long forms, in contrast to the 55 and 28 kDa biotinylated polypeptides synthesized by the short epimastigotes. Additionally, flow cytometry analysis showed that the short forms had relatively lower biotin surface binding than long ones. The involvement of phosphatases with the trypanosomatid differentiation has been proposed. In this sense, T. rangeli living parasites were able to hydrolyze the artificial substrate p-nitrophenylphosphate at a rate of 25.57+/-2.03 and 10.09+/-0.93 nmol p-NPP x h(-1) x 10(7) cells for the short and long epimastigotes, respectively. These phosphatase activities were linear with time for at least 60 min and the optimum pH lies in the acid range. Classical inhibitors of acid phosphatases, such as ammonium molybdate, sodium fluoride, and zinc chloride, showed a significant decrease in these phosphatase activities, with different patterns of inhibition. Additionally, these phosphatase activities presented different kinetic parameters (Km and Vmax) and distinct sensitivities to divalent cations. Both epimastigotes were unable to release phosphatase to the extracellular environment. Cytochemical analysis demonstrated the localization of these enzymes on the parasite surfaces (cell body and flagellum) and in intracellular vacuoles, resembling acidocalcisomes.