Coexistence of antibodies to tick-borne agents of babesiosis and Lyme borreliosis in patients from Cotia county,State of São Paulo,Brazil

This paper reports a case of coinfection caused by pathogens of Lyme disease and babesiosis in brothers. This was the first case of borreliosis in Brazil, acquired in Cotia County, State of S o Paulo, Brazil. Both children had tick bite history, presented erythema migrans, fever, arthralgia, mialgia, and developed positive serology (ELISA and Western-blotting) directed to Borrelia burgdorferi G 39/40 and Babesia bovis antigens, mainly of IgM class antibodies, suggestive of acute disease. Also, high frequencies of antibodies to B. bovis was observed in a group of 59 Brazilian patients with Lyme borreliosis (25.4%), when compared with that obtained in a normal control group (10.2%) (chi-square = 5.6; p < 0.05). Interestingly, both children presented the highest titers for IgM antibodies directed to both infective diseases, among all patients with Lyme borreliosis.     

Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins

Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.     

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.     

Clinical relevance and virulence factors of pigmented Serratia marcescens

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD(50)=10(7) bacteria ml(-1)) and showed virulence factors, but were isolated with low frequency (3. 4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.     

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.     

Fine-mapping of the type 1 diabetes locus (IDDM4) on chromosome 11q and evaluation of two candidate genes (FADD and GALN) by affected sibpair and linkage-disequilibrium analyses

Previous studies have identified a susceptibility region for insulin-dependent (type 1) diabetes mellitus on chromosome 11q13 (IDDM4). In this study, 15 polymorphic markers were analyzed for 382 affected sibpair (ASP) families with type 1 diabetes. Our analyses provided additional evidence for linkage for IDDM4 (a peak LOD score of 3.4 at D11S913). The markers with strong linkage evidence are located within an interval of approximately 6 cM between D11S4205 and GALN. We also identified polymorphisms in two candidate genes, Fas-associated death domain protein (FADD) and galanin (GALN). Analyses of the data by transmission/disequilibrium test (TDT) and extended TDT (ETDT) did not provide any evidence for association/linkage with these candidate genes. However, ETDT did reveal significant association/linkage with the marker D11S987 (P=0.0004) within the IDDM4 interval defined by ASP analyses, suggesting that IDDM4 may be in the close proximity of D11S987.     

Bottlenecks and multiple introductions: population genetics of the vector of avian malaria in Hawaii

Avian malaria has had a profound impact on the demographics and behaviour of Hawaiian forest birds since its vector, Culex quinquefasciatus the southern house mosquito, was first introduced to Hawaii around 1830. In order to understand the dynamics of the disease in Hawaii and gain insights into the evolution of vector-mediated parasite-host interactions in general we studied the population genetics of Cx. quinquefasciatus in the Hawaiian Islands. We used both microsatellite and mitochondrial loci. Not surprisingly we found that mosquitoes in Midway, a small island in the Western group, are quite distinct from the populations in the main Hawaiian Islands. However, we also found that in general mosquito populations are relatively isolated even among the main islands, in particular between Hawaii (the Big Island) and the remaining Hawaiian Islands. We found evidence of bottlenecks among populations within the Big Island and an excess of alleles in Maui, the site of the original introduction. The mitochondrial diversity was typically low but higher than expected. The current distribution of mitochondrial haplotypes combined with the microsatellite information lead us to conclude that there have been several introductions and to speculate on some processes that may be responsible for the current population genetics of vectors of avian malaria in Hawaii.     

Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.     

In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith,an endemic bromeliad from Eastern Brazil

An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 μM BA and 2.5 μM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 μM BA combined with 2.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi

Aims:  To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different β-glucosidases-producing filamentous fungi to enhance their antioxidant activity.
Methods and Results:  Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger , Aspergillus niveus and Aspergillus awamori . The fungi studied produced approximately the same β-glucosidase activity units amount when p- nitrophenyl-β- d -glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-β- d -glucopyranoside as substrate, showed that β-glucosidase produced by A.   niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus . Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H₂O₂ generated in vivo by the tumour promoter 12- O- tetradecanoyl phorbol-13-acetate.
Conclusions:  A.   niveus synthesized a β-glucosidase with higher specificity to hydrolyse genistin β-glycosidic bond than those produced by A .  awamori and A. niger .
Significance and Impact of the Study:  The utilization of these β-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.