Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the ß-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Normal median near nerve potential

In routine studies of sensory nerve conduction, only fibers e7 µm in diameter are analyzed. The late components which originate from thinner fibers are not detected. This explains why a normal sensory action potential (SAP) may be recorded in patients with peripheral neuropathies and sensory loss. In the present study we investigated the late component of the median SAP with a near nerve needle electrode technique in 14 normal volunteers (7 men and 7 women), aged 34.5 ± 14.8 years. The stimulus consisted of rectangular pulses of 0.2-ms duration at a frequency of 1 Hz with an intensity at least 6 times greater than the threshold value for the main component. Five hundred to 2000 sweep averagings were performed. The duration of analysis was 40 or 50 ms and the wave analysis frequency was 200 (-6 dB/oct) to 3000 Hz (-12 dB/oct). We used an apparatus with a two-channel amplifier system, 200 MW or more of entry impedance and a noise level of 0.7 µVrms or less. The main component mean amplitude, conduction velocity and latency and the late component mean amplitude, conduction velocity and latency were respectively (mean ± SD): 26.5 ± 5.42 µV, 56.8 ± 5.42 m/s, 3.01 ± 0.31 ms, 0.12 ± 0.04 µV, 16.4 ± 2.95 m/s and 10.6 ± 2.48 ms. More sophisticated equipment has an internal noise of 0.6 µVrms. These data demonstrate that the technique can now be employed to study thin fiber neuropathies, like in leprosy, using commercial electromyographs, even in non-academic practices

Genome features of Leptospira interrogans serovar Copenhageni

We report novel features of the genome sequence of Leptospira interrogans serovar Copenhageni, a highly invasive spirochete. Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in mammals. Genomic sequence analysis reveals the presence of a competent transport system with 13 families of genes encoding for major transporters including a three-member component efflux system compatible with the long-term survival of this organism. The leptospiral genome contains a broad array of genes encoding regulatory system, signal transduction and methyl-accepting chemotaxis proteins, reflecting the organism's ability to respond to diverse environmental stimuli. The identification of a complete set of genes encoding the enzymes for the cobalamin biosynthetic pathway and the novel coding genes related to lipopolysaccharide biosynthesis should bring new light to the study of Leptospira physiology. Genes related to toxins, lipoproteins and several surface-exposed proteins may facilitate a better understanding of the Leptospira pathogenesis and may serve as potential candidates for vaccine.

Preparative separation and purification of sesquiterpenoids from tussilago farfara l. by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied for separation and purification of sesquiterpenoids from an extract of Tussilago farfara L. with a two-phase solvent system composed of n-hexane-ethyl acetate- methanol-water (1:0.5:1.1:0.3, v/v/v/v). The separation produced a total of 32 mg of tussilagone, 18 mg of 14-acetoxy-7β-(3'-ethyl cis-crotonoyloxy)-lα-(2'-methyl butyryloxy)-notonipetranone and 21 mg of 7β-(3'-ethyl cis-crotonoyloxy)-lα-(2'- methyl butyryloxy)-3,14-dehydro-Z-notonipetranone from 500 mg of the crude extract in one step separation with the purity of 99.5, 99.4 and 99.1%, respectively, as determined by HPLC. The structures of these compounds were identified by ESI-MS, ¹H-NMR and 13C-NMR.

Extraction and isolation of dictamnine, obacunone and fraxinellone from Dictamnus dasycarpus Turcz. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction was used to extract active compounds from the Chinese traditional medicinal D. dasycarpus under the pressure of 30 MPa and temperature of 45 ºC. Further separation and purification was established by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.8:1.3:0.9, volume ratio). The separation yielded a total of 47 mg of dictamnine, 24 mg of obacunone and 83 mg of fraxinellone from 1.0 g of the crude extract in one step separation with the purity of 99.2, 98.4 and 99.0%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by ESI-MS, IR, ¹H-NMR and 13C-NMR.

Electron paramagnetic resonance (EPR) spectral components of spin-labeled lipids in saturated phospholipid bilayers: effect of cholesterol

Electron paramagnetic resonance (EPR) spectroscopy was used to study the main structural accommodations of spin labels in bilayers of saturated phosphatidylcholines with acyl chain lengths ranging from 16 to 22 carbon atoms. EPR spectra allowed the identification of two distinct spectral components in thermodynamic equilibrium at temperatures below and above the main phase transition. An accurate analysis of EPR spectra, using two fitting programs, enabled determination of the thermodynamic profile for these major probe accommodations. Focusing the analysis on two-component EPR spectra of a spin-labeled lipid, the influence of 40 mol % cholesterol in DPPC was studied.

Preparative separation of C19-diterpenoid alkaloids from Aconitum carmichaelii Debx by pH‑zone-refining counter-current chromatography

The technique of pH-zone-refining counter-current chromatography was successfully applied to preparatively separate three C19-diterpenoid alkaloids from the crude extracts of Aconitum carmichaelii for the first time using a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v). Mesaconitine (I), hypaconitine (II), and deoxyaconitine (III) were obtained from 2.5 g of the crude alkaloids in a one-step separation; the yields were 4.16%, 16.96%, and 5.05%, respectively. The purities of compounds I, II, and III were 93.0%, 95%, and 96%, respectively, as determined by HPLC. The chemical structures of the three compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and NMR.

Separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography

An effective method for the rapid separation and purification of three stilbenes from the radix of Polygonum cillinerve (Nakai) Ohwl by macroporous resin column chromatography combined with high-speed counter-current chromatography (HSCCC) was successfully established. In the present study, a two-phase solvent system composed of chloroform-n-butanol-methanol-water (4:1:4:2, v/v/v/v) was used for HSCCC separation. A one-step separation in 4 h from 150 mg of crude extract produced 26.3 mg of trans-resveratrol-3-O-glucoside, 42.0 mg of pieceid-2"-O-gallate, and 17.9 mg of trans-resveratrol with purities of 99.1%, 97.8%, and 99.4%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these compounds were identified by nuclear magnetic resonance (NMR) spectroscopy.

Toxocariasis of the central nervous system: with report of two cases

Clinical involvement of the nervous system in visceral larva migrans due to Toxocara is rare, although in experimental animals the larvae frequently migrate to the brain. A review of the literature from the early 50's to date found 29 cases of brain involvement in toxocariasis. In 20 cases, various clinical and laboratory manifestations of eosinophilic meningitis, encephalitis, myelitis or radiculopathy were reported. We report two children with neurological manifestations, in which there was cerebrospinal fluid pleocytosis with marked eosinophilia and a positive serology for Toxocara both in serum and CSF. Serology for Schistosoma mansoni, Cysticercus cellulosae, Toxoplasma and cytomegalovirus were negative in CSF, that was sterile in both cases. Improvement of signs and symptoms after specific treatment (albendazole or thiabendazole) was observed in the two cases. A summary of data described in the 25 cases previously reported is presented and we conclude that in cases of encephalitis and myelitis with cerebrospinal fluid pleocytosis and eosinophilia, parasitic infection of the central nervous system should be suspected and serology should be performed to establish the correct diagnosis and treatment.

Root system distribution of sugar cane as related to nitrogen fertilization, evaluated by two methods: monolith and probes

Few studies on sugar cane have evaluated the root system of the crop, in spite of its importance. This is mainly due to the difficulty of evaluation and high variability of results. The objective of this study was to develop an evaluation method of the cane root system by means of probes so as to evaluate the mass, distribution and metabolically active roots related to N fertilization at planting. For this purpose, an experiment was conducted in an Arenic Kandiustults with medium texture in Jaboticabal/SP, in a randomized block design with four replications and four treatments: control (without N) and 40, 80 and 120 kg ha-1 of N applied in the form of urea in the planting furrow of the cane variety SP81 3250. One week before harvest, a urea-15N solution was applied at the cane stalk base to detect active metabolism in the root system. Trenches of 1.5 m length and 0.6 m depth were opened between two sugar cane rows for root sampling by two methods: monoliths (0.3, 0.2 and 0.15 m wide, deep and long respectively) taken from the trench wall and by probe (internal diameter 0.055 m). For each method, 15 samples per plot were collected. The roots were separated from the soil in a sieve (2 mm mesh), oven-dried (at 65 ºC) and the dry matter was measured. Root sampling by probes resulted in root mass that did not differ from the evaluation in monoliths, indicating that this evaluation method may be used for sugar cane root mass, although neither the root distribution in the soil profile nor the rhizome mass were efficiently evaluated, due to the small sample volume. Nitrogen fertilization at planting did not result in a greater root accumulation in the sugar cane plant, but caused changes in the distribution of the root system in the soil. The absence of N fertilization led to a better root distribution in the soil profile, with 50, 34 and 16 % in the 0-0.2, 0.2-0.4 and 0.4-0.6 m layers, respectively; in the fertilized treatments the roots were concentrated in the surface layer, with on average 70, 17 and 13 % for the same layers. The metabolically active roots were concentrated in the center of the cane stool, amounting to 40 % of the total root mass, regardless of N fertilization (application of 120 kg ha-1 N or without N).

Soil biological attributes in pastures of different ages in a crop-livestock integrated system

The objective of this work was to evaluate the effect of the pasture (Urochloa brizantha) component age on soil biological properties, in a crop-livestock integrated system. The experiment was carried out in a Brazilian savannah (Cerrado) area with 92 ha, divided into six pens of approximately 15 ha. Each pen represented a different stage of the pasture component: formation, P0; one year, P1; two years, P2; three years, P3; and final with 3.5 years, Pf. Samples were taken in the 0-10 cm soil depth. The soil biological parameters - microbial biomass carbon (MBC), microbial biomass respiration (C-CO2), metabolic quotient (qCO2), microbial quotient (q mic), and total organic carbon (TOC) - were evaluated and compared among different stages of the pasture, and between an adjacent area under native Cerrado and another area under degraded pasture (PCD). The MBC, q mic and TOC increased and qCO2 reduced under the different pasture stages. Compared to PCD, the pasture stages had higher MBC, q mic and TOC, and lower qCO2. The crop-livestock integrated system improved soil microbiological parameters and immobilized carbon in the soil in comparison to the degraded pasture.

Computed tomography findings of paracoccidiodomycosis in musculoskeletal system

Objective: To evaluate musculoskeletal involvement in paracoccidioidomycosis at computed tomography. Materials and Methods: Development of a retrospective study based on a review of radiologic and pathologic reports in the institution database. Patients with histopathologically confirmed musculoskeletal paracoccidioidomycosis and submitted to computed tomography were included in the present study. The imaging findings were consensually described by two radiologists. In order to avoid bias in the analysis, one patient with uncountable bone lesions was excluded from the study. Results: A total of seven patients were included in the present study. A total of 18 bone lesions were counted. The study group consisted of 7 patients. A total number of 18 bone lesions were counted. Osteoarticular lesions were the first manifestation of the disease in four patients (57.14%). Bone lesions were multiple in 42.85% of patients. Appendicular and axial skeleton were affected in 85.71% and 42.85% of cases, respectively. Bone involvement was characterized by well-demarcated osteolytic lesions. Marginal osteosclerosis was identified in 72.22% of the lesions, while lamellar periosteal reaction and soft tissue component were present in 5.55% of them. One patient showed multiple small lesions with bone sequestra. Conclusion: Paracoccidioidomycosis can be included in the differential diagnosis of either single or multiple osteolytic lesions in young patients even in the absence of a previous diagnosis of pulmonary or visceral paracoccidioidomycosis

Persistent organic pollutants in two reservoirs along the Paraíba do Sul-Guandu River system, Rio de Janeiro, Brazil

Sediment contamination is evaluated by determining organic micropollutants (organochlorine compounds - OCs and polycyclic aromatic hydrocarbons - PAHs) in two important Brazilian water reservoirs. Trace levels of OCs were observed in the Santana reservoir (44.8 ng g-1 d.w. of p,p'-DDT), while in the Funil reservoir the levels were below detection level. Forty-eight percent of the found sigmaocs were polychlorinated biphenyls, 29% dichlorodiphenyltrichloroethane (DDT), 18% Drins, and 5% other pesticides (HCB, Heptachlor, Heptachlor-epoxide, gamma-HCH and a-Endosulfan). We observed lower levels of sigmaPAH in the Funil reservoir (1 to 275 ng g-1d.w.) than in the Santana reservoir (2.2 to 26.7 µg g-1 d.w.).