Pregnane glycosides from Caralluma adscendens var. fimbriata

Eleven novel pregnane glycosides, 2-7 and 9-13, of which four, i.e., 10-13, comprised a new pregnane-type genin exhibiting a hydroxymethylene instead of a Me group at C(19), and the known pregnane glycoside stalagmoside V (8) were isolated from whole plants of Caralluma adscendens var. fimbriata, a native Indian succulent plant. Their structures were elucidated by extensive 2D-NMR spectroscopic studies.     

FGF signals from the nasal pit are necessary for normal facial morphogenesis

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.     

Conformational analysis corresponding to intra-chain disulfide bridged peptides in proteins of known three-dimensional structure

We have carried out a systematic analysis in order to evaluate whether Intra-Chain Disulfide Bridged Peptides (ICDBPs) observed in proteins of known three-dimensional structure adopt structurally similar conformations as they may correspond to structural/functional motifs. 406 representative ICDBPs comprising between 3 to 17 amino acid residues could be classified according to peptide sequence length and main-chain secondary structure conformation into 146 classes. ICDBPs comprising 6 amino acid residues are maximally represented in the Protein Data Bank. They also represent the maximum number of main-chain secondary structure conformational classes. Individual ICDBPs in each class represent different protein superfamilies and correspond to different amino acid sequences. We identified 145 ICDBP pairs that had not less-than 0.5 A root mean square deviation value corresponding to their equivalent peptide backbone atoms. We believe these ICDBPs represent structural motifs and possible candidates in order to further explore their structure/function role in the corresponding proteins. The common conformational classes observed for ICDBPs defined according to the main-chain secondary structure conformations; H (helix), B (residue in a isolated beta bridge), C (coil), E (extended beta strand), G (3(10) helix), I (pi helix), S (bend), T (hydrogen-bonded turn) were; "CHHH", "CTTC", "CSSS" and "CSSC" (for ICDBP length 4), "CSSCC" (length 5), "EETTEE", "CCSSCC", "CCSSSC" (length 6), "EETTTEE" (length 7), "EETTTTEE" (length 8), "EEEETTEEEE" (length 10), "EEEETTTEEEE" (length 11) and "EEEETTTTEEEE" (length 12).     

Entomopathogenic nematodes for control of Phyllophaga georgiana (Coleoptera: Scarabaeidae) in cranberries

A series of laboratory and greenhouse experiments evaluated the entomopathogenic nematodes Steinernema scarabaei Stock & Koppenhöfer, Heterorhabditis bacteriophora Poinar, and H. zealandica Poinar for control of second- and third-instar cranberry white grub, Phyllophaga georgiana Horn (Coleoptera: Scarabaeidae), in cranberries. Steinernema scarabaei was the most effective species with 76–100% control at a rate of 2.5×10⁹ IJ/ha in the greenhouse experiments. H. zealandica and especially H. bacteriophora were generally less effective and required rates of 5×10⁹ IJ/ha for acceptable control. Larval stage had no effect on H. zealandica and H. bacteriophora performance, whereas S. scarabaei was more effective against third instars than second instars in the laboratory but not in the greenhouse experiments. Steinernema scarabaei, should it become commercially available, could be an effective alternative to chemical insecticides for P. georgiana management.     

Carbon-hydrogen and carbon-heteroatom bond activation using iridium(I) complexes

In this research, the B3LYP density functional and Stevens effective core potentials are used to compare carbon-hydrogen and carbon-heteroatom bond activation by an iridium(I) complex. Of particular importance is to address the kinetic (transition state) and thermodynamic (ground state) selectivity. The complex Ir(PH₃)₂(H) with CH₃-X (X=F, Cl, OH, SH, NH₂, PH₂) as the substrate has been used as a model. Good agreement in geometries is obtained between the target molecules and experimental models. The resultant products of C-H and C-X oxidative addition are Y-shaped minima (i.e., a distorted trigonal bipyramid with one acute and two obtuse angles among the equatorial ligands). Oxidative addition of the C-X bond to the substrate is exothermic for groups 16 and 17, but endothermic for group 15. A significant thermodynamic preference for C-X activation over C-H activation is observed for these Ir(I) complexes. However, analysis of the transition states for oxidative addition suggests that there is a kinetic preference for C-H activation.     

Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific protein-protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.     

Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.