Selective killing of nonreplicating mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.     

Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin (AA-5-HT), and the endocannabinoid reuptake inhibitor, OMDM-2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2-arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender-dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.     

Hematologic Values of Free-ranging Cebus cay and Cebus nigritus in Southern Brazil

We evaluated blood samples obtained from 80 free-ranging healthy capuchins (Cebus cay and C. nigritus) to establish hematological reference values and to assess the influence of sex and age on them. We caught the monkeys in the Paraná River region of Southern Brazil via manual or automatic traps. We anesthetized them intramuscularly with 3.6 mg/kg tiletamine/zolazepam hydrochlorides. After physical examinations, we divided the sample according to sex and age: 26 females (13 adults and 13 juveniles) and 54 males (27 adults and 27 juveniles). We collected blood and determined hematological values via traditional published methods. We analyzed data via 2-way ANOVA to test the effect of sex, age, and interactions between the 2 factors. The packed-cell volume was higher in adult males and the numbers of white blood cells and lymphocytes were higher in juveniles. There is no other significant difference.     

Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development

Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.     

Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

Metabolic engineering for bioproduction of sugar alcohols

Sugar alcohols find applications in pharmaceuticals, oral and personal care products, and as intermediates in chemical synthesis. While industrial-scale production of these compounds has generally involved catalytic hydrogenation of sugars, microbial-based processes receive increasing attention. The past few years have seen a variety of interesting metabolic engineering efforts to improve the capabilities of bacteria and yeasts to overproduce xylitol, mannitol, and sorbitol. Examples include heterologous expression of yeast xylose reductase in Escherichia coli for the production of xylitol, coexpression of formate dehydrogenase, mannitol dehydrogenase, and a glucose facilitator protein in Corynebacterium glutamicum for mannitol production from fructose and formate, and overexpression of sorbitol-6-phosphate dehydrogenase in lactate dehydrogenase-deficient Lactobacillus plantarum to achieve nearly maximum theoretical yields of sorbitol from glucose.     

Partial purification and characterization of a non-specific acid phosphatase in leaves and root nodules of Phaseolus vulgaris.

Acid phosphatase (ACP) activity in common bean grown with or without 1.5 mM of phosphate has been examined. Leaves and root nodules responded to the absence of an exogenous phosphate source with an increase in ACP activity. Increases in enzyme activity were not associated with the synthesis of new isoforms of the enzyme. We partially purified and characterized the ACPs, which consisted of three proteins, one of leaf and two of nodule. Proteins of leaf migrated at 72 and 51 kDa in SDS-PAGE, whereas that of nodule migrated at 72, 49, 41 and 34 kDa. Enzymes of both organs had a pH optimum of 5.6, and were relatively heat stable. The enzymes exhibit a broad substrate selectivity, with maximal activity obtained with alpha-naphthyl-phosphate, ribulose 1,5-bisphosphate and p-nitrophenyl-phosphate (p-NPP). Potent inhibition by Zn2+, Hg2+, Cu2+, Pb2+, Al3+ and (MoO4)2- was observed.