Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.     

Quantitative trait loci for growth trajectories in Populus

Growth trajectories are a biological process important to plant and animal breeding, and to evolutionary genetic studies. In this article, we report the detection of quantitative trait loci (QTLs) responsible for growth trajectories in poplars that are used as a model system for the study of forest biology. These QTLs were localized on a genetic linkage map of polymorphic markers using a statistical mapping method incorporating growth-curve models. The effects of the QTLs on growth are described as a function of age, so that age-specific changes in QTL effects can be readily projected throughout the entire growth process. The QTLs identified display increased effects on growth when trees age, yet the timing of QTL activation is earlier for stem height than diameter, which is consistent with the ecological viewpoint of canopy competition. The implications of the results for breeding and silviculture are discussed.     

Ontogenetic characteristics of the vomeronasal organ in Saguinus geoffroyi and Leontopithecus rosalia,with comparisons to other primates

It has been suggested that the variability of the primate vomeronasal organ (VNO) may be greater than previously thought, especially among New World monkeys. It is not clear to what extent VNO variation reflects ontogenetic, functional, or phylogenetic differences among primates. The present study investigated VNO anatomy in an ontogenetic series of two genera of callitrichid primates, in order to assess recent attempts to develop VNO character states and to examine the evidence for VNO functionality at different life stages. A sample of six Leontopithecus rosalia, one L. chrysomelas, and six Saguinus geoffroyi was serially sectioned and stained using various methods. Two adult Callithrix jacchus were also sectioned for comparative purposes. The VNO of each primate was examined by light microscopy along its entire rostrocaudal extent. VNOs of the tamarins were described to determine whether they fit into 1 of 3 character states recently attributed to various New World monkeys. At birth, the two species of tamarins differed in the nature of communication between the VNO and nasopalatine duct (NPD). Two of 3 neonatal S. geoffroyi exhibited a fused VNO duct in a more dorsal position (adjacent to the nasal cavity) compared to that of L. rosalia. The VNO duct communicated with the NPD and was patent in neonatal L. rosalia. Both species appeared to have an age-related increase in the amount of sensory epithelium in the VNO. Subadult L. rosalia had caudal regions of the VNO that were exceptionally well-developed, similar to those of strepsirhine primates. Compared to subadults, all adult callitrichids appeared to have more ventral communications of the VNO duct directly into the NPD. Adult S. geoffroyi and L. chrysomelas both had VNO sensory epithelium separated by multiple patches of nonsensory epithelium. This contrasted with the VNOs of C. jacchus, which had a nearly continuous distribution of receptors on all surfaces of the VNO. The findings indicate that tamarins have delayed maturation of the VNO epithelium, and that some species have little or no perinatal function. These results also suggest that ontogenetic changes in craniofacial form may alter the position of the VNO in tamarins. The present study supports the use of at least two character states to categorize the VNO of various callitrichids, but it is suggested that one of these, previously called "reduced sensory epithelium" should be instead termed "interrupted sensory epithelium." The distribution of VNO sensory epithelium does not appear to reflect phylogenetic influences; it is more likely a functional characteristic that varies throughout postnatal life. Therefore, this chemosensory system has a high degree of plasticity relating to age and function, which in some instances can confound the use of characteristics as phylogenetic traits. Further study is needed to quantify VNO receptors in various species to determine if functional differences exist and if some species have more precocious VNO function than others.     

The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival

In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second, the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4(-/-) embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence.     

Chemo- and stereoselective synthesis of benzocycloheptene and 1-benzoxepin derivatives as alpha-sympathomimetic and anorexigenic agents

The synthesis and pharmacological evaluation of cis- and trans-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ols 4a-c and 5a-c and cis- and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ols 4d-f and 5d-f were carried out. Chemo- and stereoselective synthesis of 5a-f was achieved by reduction of corresponding alpha-amino ketones 3a-f with LiAl(t-BuO)3H. cis-4-Amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 4d and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 5d exhibited marked anorexigenic activity in mice at a dose of LD50 800 and 500 mg/kg and ED50 75 and 55 mg/kg, respectively, while the analog cis-2,3-dihydroxy-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol 8 showed typical alpha-sympathomimetic activity.     

Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax

The bacterial storage polymer poly-β-hydroxybutyrate (PHB) has the potential to be used as an alternative anti-infective strategy for aquaculture rearing. In this research, the effects of (partially) replacing the feed of European sea bass juveniles with PHB were investigated. During a 6-week trial period, the PHB showed the ability to act as an energy source for the fish. This indicated that PHB was degraded and used during gastrointestinal passage. The gut pH decreased from 7.7 to 7.2 suggesting that the presence of PHB in the gut led to the increased production of (short-chain fatty) acids. The diets supplemented with 2% and 5% PHB (w/w) induced a gain of the initial fish weight with a factor 2.4 and 2.7, respectively, relative to a factor 2.2 in the normal feed treatment. Simultaneously, these treatments showed the highest bacterial range-weighted richness in the fish intestine. Based on molecular analysis, higher dietary PHB levels induced larger changes in the bacterial community composition. From our results, it seems that PHB can have a beneficial effect on fish growth performance and that the intestinal bacterial community structure may be closely related to this phenomenon.     

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ? H of curcumin heptadiene chain are closely positioned to the A₁₆-H8 and A₁₇-H8, while G₁₂-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Development of the nasolacrimal apparatus in the Mongolian gerbil (Meriones unguiculatus), with notes on network topology and function

The nasolacrimal apparatus (NLA) is a multicomponent functional system comprised of multiple orbital glands (up to four larger multicellular exocrine structures), a nasal chemosensory structure (vomeronasal organ: VNO), and a connecting duct (nasolacrimal duct: NLD). Although this system has been described in all tetrapod vertebrate lineages, albeit not always with all three main components present, considerably less is known about its ontogeny. The Mongolian gerbil (Meriones unguiculatus) is a common lab rodent in which the individual components of the adult NLA have been well studied, but as yet nothing is known about the ontogeny of the NLA. In this study, serial sections of 15 fetal and three adult Mongolian gerbil heads show that the development of the NLA falls into three fetal stages: inception (origin of all features), elongation (lengthening of all features), and expansion (widening of all features). No postnatal or juvenile specimens were observed in this study, but considerable growth evidently occurs before the final adult condition is reached. The development of the orbital glands and the VNO in the Mongolian gerbil is largely consistent with those in other mammals, despite a slight nomenclatural conundrum for the anterior orbital glands. However, the Mongolian gerbil NLD follows a more circuitous route than in other tetrapods, due mainly to the convoluted arrangement of the narial cartilages, the development of a pair of enlarged incisors as well as an enlarged infraorbital foramen. The impact of these associated features on the ontogeny and phylogeny of the NLA could be examined through the approach of network science. This approach allows for the incorporation of adaptations to specific lifestyles as potential explanations for the variation observed in the NLA across different tetrapod clades. J. Morphol. 276:1005–1024, 2015. © 2015 Wiley Periodicals, Inc.