Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.     

The opposite effects of short- and long-term salt loading on pituitary adrenal axis activity in rats.

Osmotic stimulation has been shown to modify corticotropin responsiveness. We compared the effects of short- and long-term salt loading on pituitary-adrenal activity in control rats receiving tap water and rats submitted to salt loading for 1 day (S1) or 8 days (S8). Corticosterone (B) and adrenocorticotropic hormone (ACTH) plasma levels were determined at 8 a. m. under basal conditions or after immobilization stress for 15 min or corticotropin-releasing hormone (CRH) stimulation. S1 rats showed a similar ACTH response to immobilization, but an increased CRH response. In contrast, S8 rats showed blunted responses after immobilization or CRH stimulation. To evaluate the circadian variation of this inhibitory effect on the stress in the S8 group, immobilization was also performed at 8 p. m. Plasma ACTH and B levels under resting conditions were higher at 8 p. m. than 8 a. m. (p < 0.05) in control and S8 rats. The ACTH response to immobilization in the S8 group was lower than control at both 8 a. m. and 8 p. m. (p < 0.05); however, this reduction was more evident the morning, resulting in an inversion of the diurnal pattern with a higher ACTH response at 8 p. m. In conclusion, short osmotic stimulation results in an increased pituitary response to CRH, whereas prolonged stimulation decreases the pituitary response to CRH and immobilization, showing an interaction between osmoregulation and hypothalamus-pituitary-adrenocortical activity.     

Scavenger receptor class B type I is expressed in cultured keratinocytes and epidermis. Regulation in response to changes in cholesterol homeostasis and barrier requirements.

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [(3)H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.     

A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors

6-Methyl-3-phenylcoumarins 3–6 were designed, synthesized and evaluated as monoamine oxidase A and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol–coumarin hybrids) was carried out with good yield by a Perkin reaction, from the 5-methylsalicylaldehyde and the corresponding phenylacetic acid. They show high selectivity to the MAO-B isoenzyme, with IC₅₀ values in the nanomolar range. Compound 5 is the most active compound and is several times more potent and selective than the reference compound, R-(?)-deprenyl.     

Comparison between Cultured Small-Intestinal and Fecal Microbiotas in Beagle Dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10² to 10⁶ CFU/g) than in feces (range, 10⁸ to 10¹¹ CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.     

Genetic characterization of residual <Emphasis Type="Italic">Triatoma infestans</Emphasis> populations from Brazil by microsatellite

In spite of long-term efforts to eliminate Triatoma infestans (Klug 1834) from Brazil, residual foci still persist in the states of Bahia and Rio Grande do Sul. Data on the genetic variability and structuring of these populations are however lacking. Using nine microsatellite loci, we characterized one residual T. infestans population from Bahia and four from Rio Grande do Sul, and compared them with bugs originally from an older focus in São Paulo; 224 bugs were genotyped. The number of alleles per locus ranged from 5 to 11. Observed and expected heterozygosities per locus ranged, respectively, from 0 to 0.786 and from 0 to 0.764. Significant departures from Hardy–Weinberg equilibrium, mainly due to heterozygote deficits, were detected in all loci and in most populations. Global indices estimated by AMOVA were: Fis was 0.37; Fst was 0.28; and Fit was 0.55; overall indices with p?=?0.00 indicated substantial differentiation. Inter-population Fst ranged from 0.118 to 0.562, suggesting strong genetic structuring and little to no gene flow among populations. Intra-population Fis ranged from 0.301 to 0.307. Inbreeding was apparent in all populations except that from Bahia—which might be either linked by gene flow to nearby unsampled populations or part of a relatively large local population. The overall pattern of strong genetic structuring among pyrethroid-susceptible residual T. infestans populations suggests that their persistence is probably due to operational control failures. Detection and elimination of such residual foci is technically feasible and must become a public health priority in Brazil.     

Application of hydrotime model to predict early vigour of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under abiotic stresses

Rapeseed (Brassica napus L.) is important for edible oil production in semi-arid areas. Abiotic stresses are threatening rapeseed production in such areas. This study was conducted to find tolerant genotypes of rapeseed and to determine which traits of crop establishment is related to abiotic stress tolerance. Hydrotime model parameters were determined in a laboratory germination test, and seedling emergence and growth were evaluated in pot experiments under control, drought, salinity, deep sowing, low and high temperatures for 19 rapeseed genotypes. Results indicated that the predicted germination time courses at the various water potentials generally fitted well with the observed germination data. The estimated values of θ _H, ψ_b(50), and σψ_b differed significantly across genotypes. Seedling emergence and growth differed significantly under each environmental condition. PCA showed that genotypes of Hayola 401 and line 285 were the most tolerant to abiotic stresses during crop establishment and seedling growth. The first PC explained 40% of variations, and a correlation was observed between PC1 and ψ_b(50). Correlations among hydrotime model parameters and early seed vigour variables indicated that ψ_b(50) negatively correlated with seedling emergence percentage and rate (day^?1) under all abiotic stresses. It shows that genotypes with more negative values of ψ_b(50) have more seedling emergence percentage and a larger seedling emergence rate (days^?1) under a wide range of environmental conditions. Thus, it can be concluded that, to identify tolerant genotypes of rapesee to abiotic stresses, ψ_b(50) is a good trait and that breeders can focus on reducing ψ_b(50) to increase tolerance of abiotic stresses.     

Building proteomic tool boxes to monitor MHC class I and class II peptides

Major histocompatibility complex Class I (MHCI) and Class II (MHCII) presented peptides powerfully modulate T cell immunity and play a vital role in generating effective anti‐tumor and anti‐viral immune responses in mammals. Characterizing these MHCI or MHCII presented peptides can help generate therapeutic treatments, afford information on T cell mediated biomarkers, provide insight into disease progression, and reduce adverse anti‐drug side effects from engineered biotherapeutics. Here, we explore the tools and techniques commonly employed to discover both MHCI‐ and MHCII‐presented peptides. We describe complementary strategies that enhance the characterization of these peptides and the informatics tools employed for both predicting and characterizing MHCI‐ and MHCII‐presented epitopes. The evolution of methodologies for isolating MHC‐presented peptides is discussed, as are the mass spectrometric workflows that can be employed for their characterization. We provide a perspective on where this field is headed, and how these tools may be applicable to the discovery and monitoring of epitopes in a variety of scenarios.