Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications

Background

The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males.

Results

AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet.

Conclusions

Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

     

Risk assessment for developing gliomas: a comparison of two cytogenetic approaches

Chromosome instability (CIN) measured as chromosome aberrations has long been suggested as a cancer susceptibility biomarker. Conventional cytogenetic end-points are now being improved by combining molecular methods, which increases the sensitivity, specificity, and precision of the assay. In this study we examined both spontaneous and gamma-ray induced CIN in lymphocyte cultures from 51 previously untreated glioma patients and 51 age-, sex- and ethnicity-matched controls. CIN was assessed using two parallel methods: (1) the mutagen sensitivity (MS) assay and (2) the multicolor fluorescence in situ hybridization (FISH) assay. The frequency of spontaneous breaks was significantly higher in glioma patients (mean+/-S.D., 2.12+/-1.07) than in controls (1.24+/-0.86, P<0.001) when using the FISH assay but not the MS assay (0.019+/-0.02 and 0.019+/-0.01, respectively; P=0.915). Similarly, the frequency of induced chromatid breaks was significantly higher using the FISH assay (3.39+/-1.72) but not the MS assay (0.42+/-0.16) in the patients versus controls (2.08+/-1.18 and 0.37+/-0.15, respectively; P<0.001 and P=0.10, respectively). By using the median number of breaks in the controls as the cutoff value, we observed an odds ratio (ORs) of 5.13 (95% CI=2.23-12.1) for spontaneous and 4.86 (95% CI=2.08-11.4) for induced CIN using the FISH assay, whereas the ORs were 1.32 (95% CI=0.49-3.58) and 1.28 (95% CI=0.59-2.80) for spontaneous and induced CIN using the MS assay. There was also a significant increase in the frequency of hyperdiploid cells in the glioma cases which could only be detected using the FISH assay (OR=4.0, 95% CL=0.9-17.0). By combining both methods an estimated risk of 7.0 (95% CI=1.7-25.6) was observed. There was no correlation between the breaks detected by the two methods suggesting that each method is a measure of a different event. The results indicate that using the multicolor FISH assay for detection of CIN in peripheral blood lymphocytes in glioma patients is a more useful marker for risk assessment.     

Use of wastewater as a nutrient solution in a closed gravel hydroponic culture of giant reed (Arundo donax)

The objective of this experimental work was to examine the efficiency of giant reed (Arundo donax L.), as a source of biomass production and as a biofiltering device for sewage effluents. Two giant reed populations were cultivated in a closed gravel hydroponic system, where pig's waste was used as a nutrient solution. The results showed that stem biomass production varied from 12 to 23 kg DM m(-2) yr(-1), more than the ordinary production in the soil. According to stem analysis, for the first two years, there was an average infiltration rate of 31 g m(-2) yr(-1) total N, 7.5 g m(-2) yr(-1) total P, 18.8 g m(-2) yr(-1) K, 2.1 g m(-2) yr(-1) Ca, 2.1 g m(-2) yr(-1) Mg, 0.27 g m(-2) yr(-1) Fe, 0.02 g m(-2) yr(-1) Mn, 0.14 g m(-2) yr(-1) Zn and 0.08 g m(-2) yr(-1) Cu. During the third year, when a nutrient solution with added P was used, the average infiltration rate for most elements increased by 46% and for P by 169%.     

A scanning tunnelling microscopy study of Clostridium pasteurianum rubredoxin

Scanning tunnelling microscopy (STM), which can provide 'direct' and 'non-averaged' information on molecular structure in three dimensions, has been used to achieve sub-molecular resolution in a 'single molecule' of rubredoxin, an important iron-sulphur protein, at the gold (111)/water interface. The metal-ligand site [Fe(III)-Cys4] appears distinct because of an enhancement of the tunnelling current over this region compared to the surrounding protein structure.     

Interaction of Neuropeptides and Cultured Glial (Müller) Cells of the Chick Retina: Elevation of Intracellular Cyclic AMP by Vasoactive Intestinal Peptide and Glucagon

Vasoactive intestinal peptide (VIP) and, to a lesser extent, glucagon were found to increase intracellular cyclic AMP rapidly in cultured glial (Müller) cells of the chick embryo retina. Although VIP elicited higher cyclic AMP accumulation than glucagon at each concentration tested, the half-maximal concentrations were similar, i.e., 6 X 10(-8) M for VIP and 8 X 10(-8) M for glucagon. Secretin had a minimal effect on cyclic AMP accumulation even at a very high (5 X 10(-6) M) concentration. Several other peptide and nonpeptide putative agonists also had little effect on cyclic AMP accumulation. The cultured Müller cell may thus be a useful model for examining VIP and glucagon effects on glial elements of the CNS.     

Alteration in gene expression at the onset of human Y-79 retinoblastoma cell differentiation

We have tested the hypothesis that differentiation and growth arrest of Y-79 human retinoblastoma cells in culture is associated with a modification of gene expression. We first examined proteins translated from mRNAs isolated from Y-79 cells growing in suspension and in attachment cultures in serum-containing medium and found them to be markedly different. This suggests that membrane-substrate interactions are of major consequence in the biochemical differentiation of these cells. Secondly, we examined the patterns of proteins translated from attached cells which had been induced to morphologically differentiate into neuronal-like and glial-like cells by serum-withdrawal and dibutyryl cAMP treatment respectively. The in vitro translatable proteins of mRNAs isolated from these cultures were found to be markedly different from those of the suspension and attachment cultures. Thirdly, we found that treatment of cells growing in attachment culture in serum-containing medium supplemented with 8-bromo cAMP, butyrate and retinoic acid as well as dibutyryl cAMP resulted in discreet alterations in proteins translated in vitro from extracted mRNAs. Although all these substances inhibit the growth of Y-79 cells, only dibutyryl cAMP and butyrate result in morphological differentiation of cells. Our results suggest that (1) attachment and morphological differentiation of Y-79 cells are both related to specific alterations in gene expression and (2) differentiation and inhibition of cell growth by various agents can be correlated with changes in translatable mRNA species although all agents do not act in the same mode.     

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.     

Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101).

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W-V247L (80 min-1 or 2.5 x the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.     

Cyclic AMP and Butyrate Modulate Melatonin Synthesis in Y79 Human Retinoblastoma Cells

Abstract: Melatonin is synthesized by cultured Y79 human retinoblastoma cells and is secreted into the medium. Activity of the two key enzymes involved in the synthesis of melatonin, N -acetyltransferase (NAT) and hydroxyindole- O -methyl-transferase (HIOMT), are present in retinoblastoma cells. The activity of these enzymes and the resulting synthesis and release of melatonin are modulated by the addition of a cyclic AMP analogue and butyrate to the culture medium. Melatonin levels increase dramatically over control levels after the addition of dibutyryl cyclic AMP (dbcAMP), whereas melatonin levels decrease after butyrate treatment. HIOMT activity is inhibited by both dbcAMP and butyrate, and NAT activity is stimulated by both of these differentiating agents, suggesting that the rise in melatonin levels in response to dbcAMP is the result of increased activity of NAT, whereas the decline in melatonin levels in response to butyrate may be due to a drop in HIOMT activity. Melatonin synthesis is dose- and time-dependent, and the effect of dbcAMP is readily reversible, whereas the effect of butyrate does not appear to be reversible. These effects probably reflect basic differences in the regulatory mechanisms of the inducing agents.