Effect of magnesium chloride and guanidinum chloride on the extraction of components of extracellular matrix from chicken cartilage

In order to evaluate the effect of chaotropic agents on proteoglycan and non-collagenous proteins, chicken xiphoid cartilage was treated with guanidine-HCI and MgCl2 in different concentrations (1M to 5M), and different periods of time (12, 24, 48 and 72hr). The maximum yield of uronic acid was obtained with 3M MgCl2 (73.3 per cent). Concentrations of 4M and 5M of MgCl2 showed that much less uronic acid was removed, 55.3 per cent and 38.1 respectively. Extraction with 3M MgCl2 and 3M guanidine-HCl resulted better efficiency when performed for 48 hr. Analysis by SDS-PAGE of the extracts obtained with guanidine-HCl and MgCl, in different concentrations pointed out that most components are equally removed with the two solvents, showing that the extraction with MgCl2 is an alternative assay to remove non-collagenous proteins from extracellular matrix.

Identification of Mycobacteria by Thin Layer Chromatographic Analysis of Mycolic Acids and Conventional Biochemical Method: Four Years of Experience

Mycolic acids analysis by thin-layer chromatography (TLC) has been employed by several laboratories worldwide as a method for fast identification of mycobacteria. This method was introduced in Brazil by our laboratory in 1992 as a routine identification technique. Up to the present, 861 strains isolated were identified by mycolic acids TLC and by standard biochemical tests; 61% out of these strains came as clinical samples, 4% isolated from frogs and 35% as environmental samples. Mycobacterium tuberculosis strains identified by classical methods were confirmed by their mycolic acids contents (I, III and IV). The method allowed earlier differentiation of M. avium complex - MAC (mycolic acids I, IV and VI) from M. simiae (acids I, II and IV), both with similar biochemical properties. The method also permitted to distinguish M. fortuitum (acids I and V) from M. chelonae (acids I and II) , and to detect mixed mycobacterial infections cases as M. tuberculosis with MAC and M. fortuitum with MAC. Concluding, four years experience shows that mycolic acids TLC is an easy, reliable, fast and inexpensive method, an important tool to put together conventional mycobacteria identification methods.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Effect of the Contents and Form of Rabies Glycoprotein on the Potency of Rabies Vaccination in Cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40°C, 1 h) or cold (5 or 0°C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10-14 to 10-7 M) and/or losartan, 10-16 to 10-6 M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cytotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10-13 M and 10-12 M AII concentrations. The addition of losartan (up to10-14 M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.

A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Effect of reducing sensory and environmental stimuli during hospitalized premature infant sleep

OBJECTIVETo compare the total sleep time of premature infant in the presence or absence of reducing sensory and environmental stimuli in the neonatal unit.METHODLongitudinal study in a Neonatal Intermediate Care Unit of a public hospital in Sao Paulo. The sample consisted of 13 premature infants. We used polysomnograph and unstructured observation for data collection. We analyzed 240 and 1200 minutes corresponding to the periods of the presence and absence of environmental management, respectively. Data were compared in proportion to the total sleep time in the two moments proposed by the study.RESULTSThe total sleep time in periods without environmental management was on average 696.4 (± 112.1) minutes and with management 168.5 (± 27.9) minutes, proportionally premature infant slept an average of 70.2% during periods with no intervention and 58.0% without management (p=0.002).CONCLUSIONReducing stimulation and handling of premature infant environment periods was effective to provide greater total sleep time.

Selection of artificial hosts for oviposition by wild Anastrepha obliqua (Macquart) (Diptera, Tephritidae): influence of adult food and effect of experience

Several factors influence the selection of oviposition substrates by insects. The aim of the present work was to find answers to the following questions related to the oviposition behavior of Anastrepha obliqua. Can carbohydrates (glucose or sucrose) present in the adult diet have influence on the female preference for an oviposition substrate with similar composition? Can the previous experience with a host containing one of mentioned carbohydrates interfere in further selection of oviposition substrates? The results showed that the kind of carbohydrate present in the adult diet did not affect the female preference for an artificial oviposition substrate, neither when it was presented by itself nor in combination with brewer's yeast. The effect of experience in the oviposition behavior was observed when there was a previous contact with artificial oviposition substrates containing yeast and sucrose. The data are discussed in terms of the behavioral plasticity presented by this species in relation to feeding and oviposition behaviors.

Water retention and availability in soils of the State of Santa Catarina-Brazil: effect of textural classes, soil classes and lithology

The retention and availability of water in the soil vary according to the soil characteristics and determine plant growth. Thus, the aim of this study was to evaluate water retention and availability in the soils of the State of Santa Catarina, Brazil, according to the textural class, soil class and lithology. The surface and subsurface horizons of 44 profiles were sampled in different regions of the State and different cover crops to determine field capacity, permanent wilting point, available water content, particle size, and organic matter content. Water retention and availability between the horizons were compared in a mixed model, considering the textural classes, the soil classes and lithology as fixed factors and profiles as random factors. It may be concluded that water retention is greater in silty or clayey soils and that the organic matter content is higher, especially in Humic Cambisols, Nitisols and Ferralsol developed from igneous or sedimentary rocks. Water availability is greater in loam-textured soils, with high organic matter content, especially in soils of humic character. It is lower in the sandy texture class, especially in Arenosols formed from recent alluvial deposits or in gravelly soils derived from granite. The greater water availability in the surface horizons, with more organic matter than in the subsurface layers, illustrates the importance of organic matter for water retention and availability.

Effect of management systems and cover crops on organic matter dynamics of soil under vegetables

Vegetable production in conservation tillage has increased in Brazil, with positive effects on the soil quality. Since management systems alter the quantity and quality of organic matter, this study evaluated the influence of different management systems and cover crops on the organic matter dynamics of a dystrophic Red Latosol under vegetables. The treatments consisted of the combination of three soil tillage systems: no-tillage (NT), reduced tillage (RT) and conventional tillage (CT) and of two cover crops: maize monoculture and maize-mucuna intercrop. Vegetables were grown in the winter and the cover crops in the summer for straw production. The experiment was arranged in a randomized block design with four replications. Soil samples were collected between the crop rows in three layers (0.0-0.05, 0.05-0.10, and 0.10-0.30 m) twice: in October, before planting cover crops for straw, and in July, during vegetable cultivation. The total organic carbon (TOC), microbial biomass carbon (MBC), oxidizable fractions, and the carbon fractions fulvic acid (C FA), humic acid (C HA) and humin (C HUM) were determined. The main changes in these properties occurred in the upper layers (0.0-0.05 and 0.05-0.10 m) where, in general, TOC levels were highest in NT with maize straw. The MBC levels were lowest in CT systems, indicating sensitivity to soil disturbance. Under mucuna, the levels of C HA were lower in RT than NT systems, while the C FA levels were lower in RT than CT. For vegetable production, the C HUM values were lowest in the 0.05-0.10 m layer under CT. With regard to the oxidizable fractions, the tillage systems differed only in the most labile C fractions, with higher levels in NT than CT in the 0.0-0.05 m layer in both summer and winter, with no differences between these systems in the other layers. The cabbage yield was not influenced by the soil management system, but benefited from the mulch production of the preceding maize-mucuna intercrop as cover plant.

Effect of potassium sources and rates on arabica coffee yield, nutrition, and macronutrient export

The use of potassium (K) rock powder can be an alternative for K supply of crops. Thus, to reduce K fertilizer imports from abroad, possibilities of extracting this nutrient from Brazilian rocks are being studied. The objective was to evaluate the effect of phonolite rock powder (F2) as K source (Ekosil®) on the air-dried fruit yield, nutrition and macronutrient export of Arabica coffee. The experiment was carried out on a dystroferric Red Latosol (Typic Haplorthox), in Piraju, São Paulo State, Brazil, in the 2008/09 and 2009/10 growing seasons. The experimental design was a randomized complete block, in a factorial 2 × 3 + 1 arrangement, with four replications. The treatments consisted of two K sources (KCl - 58 % of K2O and F2 - 8.42 % K2O) and three rates ½-, 1-, and 2-fold the K2O rate recommended for coffee, i.e., 75, 150, and 300 kg ha-1 of K2O), plus a control (without K application). Potassium supply increased coffee yield, regardless of the source. Application of source F2 increased coffee yield similarly to KCl at the recommended K rate for coffee (150 kg ha-1 K2O), proving efficient as K supply for coffee. Potassium application increased macronutrient export in coffee, especially in the growing season with higher yield.