Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.     

The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity

A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.     

Monkeys and toads define areas of endemism on Sulawesi

Abstract.— Ecological or geological phenomena can impose limits on geographic diversification that cause biogeographical patterns of distantly related but sympatrically occurring taxa to be similar. Concordant patterns of diversity facilitate conservation management because strategic designation of protected areas can capture complementary rather than redundant components of variation. Here we demonstrate that on the biodiverse Indonesian island of Sulawesi, seemingly idiosyncratic distributions of diversity in endemic monkeys (Macaca species) and toads (Bufo celebensis) are actually virtually identical on a fine geographic scale. It appears that range fragmentation has generated seven multi-taxon areas of genetic endemism, each of which should be targeted for conservation. Joint consideration of molecular phylogeography, morphology, and demography helps resolve apparent contradictions in paraphyletic macaque mitochondrial DNA and in undifferentiated toad morphology, and facilitates an understanding of biogeography and conservation genetics of Sulawesi fauna.     

Amino acid availability regulates S6K1 and protein synthesis in avian insulin-insensitive QM7 myoblasts

The regulation of S6K1 by nutritional status and insulin has been recently reported in vivo in chicken muscle despite the relative insulin resistance of this tissue as estimated by phosphatidylinositol 3-kinase (PI3-kinase) activity. The present work aimed to study the impact of amino acids on S6K1 activity in quail muscle (QM7) myoblasts. Firstly, we characterized S6K1 in QM7 cells and demonstrated the absence of insulin receptors in these cells. Secondly, we showed that amino acids in the absence of insulin induced S6K1 phosphorylation on Thr389 and concomitantly increased its enzymatic activity. Amino acid-induced S6K1 activation was inhibited by LY294002 (PI3-kinase inhibitor) and rapamycin (inhibitor of the mammalian target of rapamycin, mTOR), suggesting the involvement of an avian homolog of mTOR. The availability of individual amino acids (methionine or leucine) regulated S6K1 phosphorylation on Thr389 and QM7 protein synthesis. In conclusion, amino acids regulate S6K1 phosphorylation and activity in QM7 cells through the mTOR/PI3-kinase pathway in an insulin-independent manner.     

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering,analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.     

Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.     

Influence of ethylene-oxy spacer group on the activity of linezolid: synthesis of potent antibacterials possessing a thiocarbonyl group

The influence of an ethylene-oxy spacer element between the heterocycle and the aromatic ring in linezolid is reported. The introduction of such spacer group generated compounds with inferior antibacterial activity. However, the conversion of the acetamide group present in the linezolid analogues to either thiocarbamate or thioacetamide functionality restored the activity. The synthesis of linezolid analogues possessing the ethylene-oxy spacer group along with SAR studies with different heterocycles and preparation of some thiocarbonyl compounds possessing potent antibacterial property are presented.     

Oxygen free radical scavenger enzyme polymorphisms in systemic sclerosis

We performed a case-control study of polymorphisms of glutathione S-transferase (GST) isoenzymes and manganese superoxide dismutase (MnSOD) in black South Africans with systemic sclerosis (SSc). The frequency of the GSTM1*B phenotype was significantly decreased in the overall SSc group compared with controls (OR=0.19, p(corr)<.05), implying a possible protective effect against development of the disease. There was also a trend toward increased MnSODAla allele and phenotype frequencies in the diffuse cutaneous SSc subset compared with controls (OR=2.11 and 3.15, respectively, p(corr)<.1). Our findings provide new data on the distribution of GST and MnSOD polymorphisms in healthy Africans and further evidence that genetic factors may have a contributory role to play in predisposing to oxidative stress in SSc.