Unusually strong H-bonding to the heme ligand and fast geminate recombination dynamics of the carbon monoxide complex of Bacillus subtilis truncated hemoglobin

The active site of the oxygen-avid truncated hemoglobin from Bacillus subtilis has been characterized by infrared absorption and resonance Raman spectroscopies, and the dynamics of CO rebinding after photolysis has been investigated by picosecond transient absorption spectroscopy. Resonance Raman experiments on the CO bound adduct revealed the presence of two Fe-CO stretching bands at 545 and 520 cm-1, respectively. Accordingly, two C-O stretching bands at 1924 and 1888 cm-1 were observed in infrared absorption and resonance Raman measurements. The very low C-O stretching frequency at 1888 cm-1 (corresponding to the extremely high RR stretching frequency at 545 cm-1) indicates unusually strong hydrogen bonding between CO and distal residues. On the basis of a comparison with other truncated hemoglobin it is envisaged that the two CO conformers are determined by specific interactions with the TrpG8 and TyrB10 residues. Mutation of TrpG8 to Leu deeply alters the hydrogen-bonding network giving rise mainly to a CO conformer characterized by a Fe-CO stretching band at 489 cm-1 and a CO stretching band at 1958 cm-1. Picosecond laser photolysis experiments carried out on the CO bound adduct revealed dynamical processes that take place within a few nanoseconds after photolysis. Picosecond dynamics is largely dominated by CO geminate rebinding and is consistent with strong H-bonding contributions of TyrB10 and TrpG8 to ligand stabilization.     

Aggregation Factor as an Inhibitor of Bacterial Binding to Gut Mucosa

Modern research in the area of probiotics is largely devoted to discovering factors that promote the adherence of probiotic candidates to host mucosal surfaces. The aim of the present study was to test the role of aggregation factor (AggL) and mucin-binding protein (MbpL) from Lactococcus sp. in adhesion to gastrointestinal mucosa. In vitro, ex vivo, and in vivo experiments in rats were used to assess the adhesive potential of these two proteins expressed in heterologous host Lactobacillus salivarius BGHO1. Although there was no influence of MbpL protein expression on BGHO1 adhesion to gut mucosa, expression of AggL had a negative effect on BGHO1 binding to ileal and colonic rat mucosa, as well as to human HT29-MTX cells and porcine gastric mucin in vitro. Because AggL did not decrease the adhesion of bacteria to intestinal fragments in ex vivo tests, where peristaltic simulation conditions were missing, we propose that intestinal motility could be a crucial force for eliminating aggregation-factor-bearing bacteria. Bacterial strains expressing aggregation factor could facilitate the removal of pathogens through the coaggregation mechanism, thus balancing gut microbial ecosystems in people affected by intestinal bacteria overgrowth.     

Synchronous global assessment of gene and protein expression in colorectal cancer progression

Well-established models of colorectal cancer progression are based on the idea that the disease evolves through a multistep process involving sequential genetic mutations, suggesting that progression through clinically defined stages should correlate with well-defined patterns of gene expression. The majority of studies to date, however, have assessed these processes one gene and one protein at a time. We report the first comprehensive assessment of both gene and protein expression performed in parallel across progressive stages of human colorectal neoplasia. Remarkably, despite the global nature of the gene expression assessment, very few genes could be linked with certainty to specific proteins through currently available annotations. Furthermore, the correlation of expression between identified genes and proteins was poor. Nevertheless, both produced expression signatures that differentiated normal mucosa and nonmalignant adenomas from each other and from the malignant carcinomas and both produced fairly consistent subclasses of the malignancies, suggesting that a molecular staging might be more appropriate provided that these profiles can be tied to clinical outcome. This is potentially important as clinical staging is widely used as a prognostic indicator and used in the decision to pursue adjuvant therapies.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Acute and subchronic co-administrations to cadmium,diazinon and selenium induce apparent osteoporotic symptoms in adult male rats

Cadmium (Cd) and diazinon (DZN) are known to be environmental risk factors for various bone diseases including osteoporosis. Selenium (Se), an essential constituent of many antioxidant enzymes, has in higher concentrations negative effects on the bone. The present study was aimed to investigate possible changes in femoral bone of adult male rats after their acute and subchronic exposures to Cd, DZN and Se. A total of 30 male Wistar rats were randomized into three experimental groups. The rats in the group A (4-months-old) were injected intraperitoneally with a mixture of 2 mg CdCl₂ kg^?1, 20 mg DZN kg^?1 and 2 mg Na₂SeO₃ kg^?1 body weight and killed 36 h after xenobiotics had been injected. In the group B, young males (1-month-old) were administered with a combination of 30 mg CdCl₂ L^?1, 40 mg DZN L^?1 and 5 mg Na₂SeO₃ L^?1 in their drinking water, for 90 days. Ten 4-months-old males without toxicant supplementation served as a control group (C). After treatment period, detailed histological analysis of femoral bone was performed in each group. Our results revealed apparent osteoporotic symptoms (resorption lacunae, osteoporotic fractures) in rats from groups A and B. Moreover, histomorphometrical evaluation showed reduced bone vascularization (constricted primary osteons’ vascular canals and Haversian canals) and weakness mechanical properties of bones (smaller size of the secondary osteons) in these rats in comparison with those of the control group. Our study demonstrates for the first time that acute and subchronic co-administrations to Cd, DZN and Se induce evident manifestation characteristics of osteoporosis in male rats.     

Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa

The objective of this research was to evaluate Trichoderma harzianum strain T22 as a biocontrol agent of collar and root rot caused by different Calonectria pauciramosa isolates. Thus, the microsclerotia-forming ability and virulence of twenty C. pauciramosa isolates were assessed. Microsclerotia production varied partially among the isolates and dual culture with T22 on carnation leaf agar revealed isolates with both high and low microsclerotia-forming ability. Inoculation tests on red clover (Triflolium pratense) demonstrated its susceptibility to the pathogen. On red clover, the degree of virulence and T22 effects in controlling infections were highly variable among the isolates tested. A nursery trial performed on Feijoa sellowiana seedlings confirmed previous results, clearly indicating virulence variability among C. pauciramosa isolates. For three isolates tested in nursery trial, T22 effectiveness in controlling infection was inversely related to their degree of virulence. Overall, T. harzianum strain T22 showed good antagonist activity in reducing microsclerotia production on carnation leaf and the incidence and severity of collar and root rot on both selected hosts. This data could be crucial in developing integrated pest management strategies in ornamental plant nurseries.     

BTEX removal in a horizontal-flow anaerobic immobilized biomass reactor under denitrifying conditions

Because benzene, toluene, ethylbenzene, and xylenes (BTEX) and ethanol are important contaminants present in Brazilian gasoline, it is essential to develop technology that can be used in the bioremediation of gasoline-contaminated aquifers. This paper evaluates the performance of a horizontal-flow anaerobic immobilized biomass (HAIB) reactor fed with water containing gasoline constituents under denitrifying conditions. Two HAIB reactors filled with polyurethane foam matrices (5 mm cubes, 23 kg/m³ density and 95 % porosity) for biomass attachment were assayed. The reactor fed with synthetic substrate containing protein, carbohydrates, sodium bicarbonate and BTEX solution in ethanol, at an Hydraulic retention time (HRT) of 13.5 h, presented hydrocarbon removal efficiencies of 99 % at the following initial concentrations: benzene 6.7 mg/L, toluene 4.9 mg/L, m-xylene and p-xylene 7.2 mg/L, ethylbenzene 3.7 mg/L, and nitrate 60 mg N/L. The HAIB reactor fed with gasoline-contaminated water at an HRT of 20 h showed hydrocarbon removal efficiencies of 96 % at the following initial concentrations: benzene, 4.9 mg/L; toluene, 7.2 mg/L; m-xylene, 3.7 mg/L; and nitrate 400 mg N/L. Microbiological observations along the length of the HAIB reactor fed with gasoline-contaminated water confirmed that in the first segment of the reactor, denitrifying metabolism predominated, whereas from the first sampling port on, the metabolism observed was predominantly methanogenic.     

Comparative spermatozoal ultrastructure and molecular analysis in dromiid crabs and their phylogenetic implications for Dromiidae and Podotremata (Decapoda: Brachyura)

We described the spermatozoal ultrastructure and conducted a molecular analysis of Dromiidae Hypoconcha parasitica, Hypoconcha arcuata, Moreiradromia antillensis and Dromia erythropus. To elucidate the relationship between the different species of this brachyuran group, we also compared the spermatozoal morphologies and phylogenetic positioning among species of Dromiidae, Dromioidea and Podotremata. Specimens were collected from the northern coast of São Paulo, Brazil and were fixed and processed followed by transmission electron microscopy and molecular analysis routines. The Dromiidae spermatozoa studied are characterized by a discoidal acrosome, with three or four concentric zones, which are centrally separated by a bilaterally capitate perforatorial chamber, with a “mushroom”-shaped apex in the Hypoconchinae and a “T-shape” in Dromiinae. Above the perforatorial chamber, there is an apical protuberance, continuous with the subopercular region and the operculum, which forms a low, centrally perforated dome. Under differential interference contrast microscopy, the spermatozoa show 3 to 4 radial arms. The spermatozoal characters in Hypoconchinae and Dromiinae do not separate these subfamilies from the Dromiidae and Dromioidea. Ultrastructural differentiation was only found between representative Dromioidea and other Podotremata. Thus, the spermiotaxonomy of these Hypoconcha, Moreiradromia and Dromia species corroborated previous morphological and molecular studies, supporting the monophyly of Dromiidae and Dynomenidae in relation to Homolidae and Latreilliidae.     

Small fish use the hypoxic pelagic zone as a refuge from predators

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Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment

Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside-5'-monophosphate, and dihydrozeatin riboside-5'-monophosphate), and N-glucosides (zeatin-N(9)-glucoside, dihydrozeatin-N(9)-glucoside, zeatin-N(7)-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N(9)-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.