Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Amaranth protein presents cholesterol-lowering effect

This study describes amaranth`s protein cholesterol-lowering effect and investigates its mechanisms hypercholesterolaemia was induced in male hamsters through diet rich in casein (300 g/kg diet) containing regular levels of cholesterol (0.5 kg/g) fed during 3 weeks. Animals were divided into three groups and fed ad libitum diets for 4 weeks containing as the sole source of protein: casein (control), amaranth protein isolate or, casein + amaranth protein isolate. Plasma concentrations of cholesterol and triacylglycerols were measured at four different points: at the beginning of the study. after hypercholesterolaemia was induced, in the first week and then at the end of the experimental diet period. The reduction of the total plasma cholesterol concentration at the end of experimental period for animals fed on diets containing amaranth protein isolate pure and with casein were 27% (P < 0.05) and 48% (P < 0.05). respectively, being the non-HDL fractions the most affected. Digestibility of protein as well as excretion of cholesterol and bile acid, were investigated as the possible mechanisms for this significant hypocholesterolaemic effect. Cholesterol excretion was related to the hypocholesterolaemia but could not explain all the observed reduction. Our findings suggest that amaranth protein has a metabolic effect on endogenous cholesterol metabolism. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of extrusion on the emulsifying properties of soybean proteins and pectin mixtures modelled by response surface methodology

The objective of this study was to apply response surface methodology to estimate the emulsifying capacity and stability of mixtures containing isolated and textured soybean proteins combined with pectin and to evaluate if the extrusion process affects these interfacial properties. A simplex-centroid design was applied to the model emulsifying activity index (EAI), average droplet size (D-[4.3]) and creaming inhibition (Cl%) of the mixtures. All models were significant and able to explain more than 86% of the variation. The high predictive capacity of the models was also confirmed. The mean values for EAI, D-[4.3] and Cl% observed in all assays were 0.173 +/- 0.015 mn, 19.2 +/- 1.0 mu m and 53.3 +/- 2.6%, respectively. No synergism was observed between the three compounds. This result can be attributed to the low soybean protein solubility at pH 6.2 (<35%). Pectin was the most important variable for improving all responses. The emulsifying capacity of the mixture increased 41% after extrusion. Our results showed that pectin could substitute or improve the emulsifying properties of the soybean proteins and that the extrusion brings additional advantage to interfacial properties of this combination. (C) 2008 Elsevier Ltd. All rights reserved.

Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and the class 1 integron-associated bla(GES-7) in Brazil

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

The use of spray drying technology to reduce bitter taste of casein hydrolysate

The utilization of protein hydrolysates in food systems is frequently hindered due to their bitterness and hygroscopicity. Spray drying technology could be an alternative for reducing these problems. The aim of this work was to reduce or to mask the casein hydrolysate bitter taste using spray drying and mixtures of gelatin and soy protein isolate (SPI) as carriers. Six formulations were studied: three with 20% of hydrolysate and 80% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%) and three with 30% of hydrolysate and 70% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%). The spray-dried formulations were evaluated by SEM, hygroscopicity, thermal behavior (DSC), dissolution, and bitter taste, by a trained sensory panel using a paired-comparison test (free samples vs. spray-dried samples); all samples were presented in powder form. SEM analysis showed mostly spherically shaped particles, with many concavities and some particles with pores. All formulations were oil and water compatible and showed lower hygroscopicity values than free casein hydrolysate. At Aw 0.83, the free hydrolysate showed Tg about 25 degrees C lower than the formulations, indicating that the formulations may be more stable at Aw >= 0.65 since the glass transition should be prevented. The sensory panel found the formulations, tasted in the powder form, to be less bitter (P < 0.05) than the free casein hydrolysate. These results indicated that spray drying of casein hydrolysate with mixtures of gelatin and SPI was successful to attenuate the bitterness of casein hydrolysate. Thus, spray drying widens the possibilities of application of casein hydrolysates. (C) 2009 Elsevier Ltd. All rights reserved.

Microencapsulation of casein hydrolysate by complex coacervation with SPI/pectin

The aim of this work was to encapsulate casein hydrolysate by complex coacervation with soybean protein isolate (SPI)/pectin. Three treatments were studied with wall material to core ratio of 1:1, 1:2 and 1:3. The samples were evaluated for morphological characteristics, moisture, hygroscopicity, solubility, hydrophobicity, surface tension, encapsulation efficiency and bitter taste with a trained sensory panel using a paired comparison test. The samples were very stable in cold water. The hydrophobicity decreased inversely with the hydrolysate content in the microcapsule. Encapsulated samples had lower hygroscopicity values than free hydrolysate. The encapsulation efficiency varied from 91.62% to 78.8%. Encapsulated samples had similar surface tension, higher values than free hydrolysate. The results of the sensory panel test considering the encapsulated samples less bitter (P < 0.05) than the free hydroly-state, showed that complex coacervation with SPI/pectin as wall material was an efficient method for microencapsulation and attenuation of the bitter taste of the hydrolysate. (C) 2009 Elsevier Ltd. All rights reserved.

Thermomechanical properties of biodegradable films based on blends of gelatin and poly(vinyl alcohol)

The aim of this work was to study the effect of the poly(vinyl alcohol) (PVA) concentration on the thermal and viscoelastic properties of films based on blends of gelatin and PVA using differential scanning calorimetry (DSC) and dynamic-mechanical analysis (DMA). One glass transition was observed between 43 and 49 degrees C on the DSC curves obtained in the first scanning of the blended films, followed by fusion of the crystalline portion between 116 and 134 degrees C. However, the DMA results showed that only the films with 10% PVA had a single peak in the tan 5 spectrum. However, when the PVA concentration was increased the dynamic mechanical spectra showed two peaks on the tan 6 curves, indicating two T(g)s. Despite this phase separation behavior the Gordon and Taylor model was successfully applied to correlate T, as a function of film composition, thus determining k = 7.47. In the DMA frequency tests, the DMA spectra showed that the storage modulus values decreased with increasing temperature. The master curves for the PVA-gelatin films were obtained applying the TTS principle (T(r) = 100 degrees C). The WLF model was thus applied allowing for the determination of the constants C(1) and C(2). The values of these constants increased with increasing PVA concentrations in the blend: C(1) = 49-66 and C(2) = 463-480. These values were used to calculate the fractional free volume of the films at the T(g) and the thermal expansion coefficient of the films above the T(g). (c) 2007 Elsevier Ltd. All rights reserved.

Mechanisms Associated with Large Daily Rainfall Events in Northeast Brazil

The mechanisms resulting in large daily rainfall events in Northeast Brazil are analyzed using data filtering to exclude periods longer than 30 days. Composites of circulation fields that include all independent events do not reveal any obvious forcing mechanisms as multiple patterns contribute to Northeast Brazil precipitation variability. To isolate coherent patterns, subsets of events are selected based on anomalies that precede the Northeast Brazil precipitation events at different locations. The results indicate that at 10 degrees S, 40 degrees W, the area of lowest annual rainfall in Brazil, precipitation occurs mainly in association with trailing midlatitude synoptic wave trains originating in either hemisphere. Closer to the equator at 5 degrees S, 37.5 degrees W, an additional convection precursor is found to the west, with a spatial structure consistent with that of a Kelvin wave. Although these two sites are located within only several hundred kilometers of each other and the midlatitude patterns that induce precipitation appear to be quite similar, the dates on which large precipitation anomalies occur at each location are almost entirely independent, pointing to separate forcing mechanisms.

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN`s subregional organization to both photic input processing and rhythmic output control.

Multipotent stem cells from umbilical cord: Cord is richer than blood!

The identification of mesenchymal stem cell ( MSC) sources that are easily obtainable is of utmost importance. Several studies have shown that MSCs could be isolated from umbilical cord (UC) units. However, the presence of MSCs in umbilical cord blood (UCB) is controversial. A possible explanation for the low efficiency of MSCs from UCB is the use of different culture conditions by independent studies. Here, we compared the efficiency in obtaining MSCs from unrelated paired UCB and UC samples harvested from the same donors. Samples were processed simultaneously, under the same culture conditions. Although MSCs from blood were obtained from only 1 of the 10 samples, we were able to isolate large amounts of multi-potent MSCs from all UC samples, which were able to originate different cell lineages. Since the routine procedure in UC banks has been to store the blood and discard other tissues, such as the cord and/or placenta, we believe our results are of immediate clinical value. Furthermore, the possibility of originating different cell lines from the UC of neonates born with genetic defects may provide new cellular research models for understanding human malformations and genetic disorders, as well as the possibility of testing the effects of different therapeutic drugs.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis the most severe deep mycosis from South America Although cell mediated immunity is considered the most efficient protective mechanism against Pb infection mechanisms of innate immunity are poorly defined Herein we investigated the interaction of the complement system with high and low virulence isolates of Pb We demonstrated that Pb18 a high virulence Pb Isolate when incubated with normal human serum (NHS) induces consumption of hemolytic complement and when immobilized promotes binding of C4b C3b and C5b-C9 Both low virulence (Pb265) and high virulence (Pb18) isolates consumed C4 C3 and mannose-binding learn (MBL) of MBL-sufficient but not of MBL-deficient serum as revealed by deposition of residual C4 C3 and MBL on immune complexes and mannan However higher complement components consumption was observed with Pb265 as compared with Pb18 The suggested relationship between low virulence and significant complement activation properties of Pb isolates was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement Conversely reactivation of attenuated Pb18 results in loss of the ability to activate complement Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway and there is an Inverse correlation between complement activating ability and Pb virulence These differences could exert an influence on Innate immunity and severity of the disease developed by infected hosts (C) 2010 Elsevier Ltd All rights reserved

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Umbilical Cord Vein

Background. Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child. Objective. To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype. Methods. Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence. Results. Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed. Conclusions. The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation.

Osteopontin involvement in granuloma formation and in the severity of Paracoccidioides brasiliensis infection

The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN(+) cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.