Amphiphilic properties of polysaccharides: dextrin chains as example.

Polysaccharide chains are usually considered to be highly hydrophilic, since they contain no obvious apolar moieties. However, it is possible for even these chains to display hydrophobic character, arising out of stereochemical constraints in the chain. We had earlier shown that linear dextrin chains display amphiphilic properties, since all the hydroxyl groups are disposed on one side or face of the chain and the hydrogens disposed on the other. We provide further evidence here for this conclusion that dextrins are amphiphilic chains. In contrast, dextrans and cellulosic chains do not display amphiphilicity. Oligosaccharides that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in a heteromolecular recognition process than as homomolecular self-aggregation.     

Coupling of lipolysis and de novo lipogenesis in brown,beige, and white adipose tissues during chronic β3-adrenergic receptor activation

Chronic activation of β3-adrenergic receptors (β3-ARs) expands the catabolic activity of both brown and white adipose tissue by engaging uncoupling protein 1 (UCP1)-dependent and UCP1-independent processes. The present work examined de novo lipogenesis (DNL) and TG/glycerol dynamics in classic brown, subcutaneous “beige,” and classic white adipose tissues during sustained β3-AR activation by CL 316,243 (CL) and also addressed the contribution of TG hydrolysis to these dynamics. CL treatment for 7 days dramatically increased DNL and TG turnover similarly in all adipose depots, despite great differences in UCP1 abundance. Increased lipid turnover was accompanied by the simultaneous upregulation of genes involved in FAS, glycerol metabolism, and FA oxidation. Inducible, adipocyte-specific deletion of adipose TG lipase (ATGL), the rate-limiting enzyme for lipolysis, demonstrates that TG hydrolysis is required for CL-induced increases in DNL, TG turnover, and mitochondrial electron transport in all depots. Interestingly, the effect of ATGL deletion on induction of specific genes involved in FA oxidation and synthesis varied among fat depots. Overall, these studies indicate that FAS and FA oxidation are tightly coupled in adipose tissues during chronic adrenergic activation, and this effect critically depends on the activity of adipocyte ATGL.     

Toxicity of methylglyoxal towards rat enterocytes and colonocytes.

Effect of methylglyoxal, a bacterial metabolic product, on protein, DNA, and RNA synthesis in rat enterocytes and colonocytes was investigated. Results showed that 1 mM methylglyoxal inhibited protein, DNA, and RNA synthesis to the extent of 65-85, 65-80, and 10-20 per cent, respectively, in villus and crypt cells and colonocytes. The inhibitory pattern was similar in these various cell types. The inhibitory effect on protein and DNA synthesis was more marked than that on RNA synthesis. Inclusion of thiol compounds up to 4 mM concentration did not protect the cells from the inhibitory effect of methylglyoxal. No alteration in the level of cellular reduced glutathione and glyoxalase enzyme activity was observed when cells were incubated with 2 mM methylglyoxal. These results suggest that the antiproliferative action of methylglyoxal on eukaryotic cells may be through the inhibition of macromolecular synthesis.     

Genetic analysis and QTL mapping of the seed hardness trait in a black common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) recombinant inbred line (RIL) population

Seed hardness trait has a profound impact on cooking time and canning quality in dry beans. This study aims to identify the unknown genetic factors and associated molecular markers to better understand and tag this trait. An F_2:7 recombinant inbred line (RIL) population was derived from a cross between the hard and soft seeded black bean parents (H68-4 and BK04-001). Eighty-five RILs and the parental lines were grown at two locations in southern Manitoba during years 2014–2016. Seed samples were harvested manually at maturity to test for seed hardness traits. The hydration capacity and stone seed count were estimated by soaking the seeds overnight at room temperature following AACC method 56-35.01. Seed samples from 2016 tests were also cooked to determine effect of seed hardness on cooking quality. For mapping of genomic regions contributing to the traits, the RIL population was genotyped using the genotype by sequencing (GBS) approach. The QTL mapping revealed that in addition to the major QTL on chromosome 7 at a genomic location previously reported to affect seed-hydration, two novel QTL with significant effects were also detected on chromosomes 1 and 2. In addition, a major QTL affecting the visual appeal of cooked bean was mapped on chromosome 4. This multi-year-site study shows that despite large environmental effects, seed hardness is an oligo-genic and highly heritable trait, which is inherited independently of the cooking quality scored as visual appeal of cooked beans. The identification of the QTLs and development of SNP markers associated with seed hardness can be applied for common bean variety improvement and genetic exploitation of these traits.     

Deciphering the crucial molecular properties of a series of Benzothiazole Hydrazone inhibitors that targets anti-apoptotic Bcl-xL protein

The Bcl-2 family proteins are the central regulators of apoptosis. Due to its predominant role in cancer progression, the Bcl-2 family proteins act as attractive therapeutic targets. Recently, molecular series of Benzothiazole Hydrazone (BH) inhibitors that exhibits drug-likeness characteristics, which selectively targets Bcl-xL have been reported. In the present study, docking was used to explore the plausible binding mode of the highly active BH inhibitor with Bcl-xL; and Molecular Dynamics (MD) simulation was applied to investigate the stability of predicted conformation over time. Furthermore, the molecular properties of the series of BH inhibitors were extensively investigated by pharmacophore based 3D-QSAR model. The docking correctly predicted the binding mode of the inhibitor inside the Bcl-xL hydrophobic groove, whereas the MD-based free energy calculation exhibited the binding strength of the complex over the time period. Furthermore, the residue decomposition analysis revealed the major energy contributing residues – F105, L108, L130, N136, and R139 – involved in complex stability. Additionally, a six-featured pharmacophore model – AAADHR.89 – was developed using the series of BH inhibitors that exhibited high survival score. The statistically significant 3D-QSAR model exhibited high correlation co-efficient (R² = .9666) and cross validation co-efficient (Q² = .9015) values obtained from PLS regression analysis. The results obtained from the current investigation might provide valuable insights for rational drug design of Bcl-xL inhibitor synthesis.     

Synthesis of enamino-2-oxindoles via conjugate addition between α-azido ketones and 3-alkenyl oxindoles: Cytotoxicity evaluation and apoptosis inducing studies

A facile method for the construction of double bond between 3-ylidene oxindoles and α-azido ketones has been successfully accomplished with a mild base. This method features azido reduction with concomitant double bond formation to provide the new class of bioactive enamino-2-oxindoles. These new compounds were screened for their in vitro cytotoxic potential on selected human cancer cell lines such as colon, lung, breast, and cervical cancer cells. Among them, representative compounds 3a, 3h, 3k, 3p, 3w and 3x showed notable cytotoxicity profile with IC₅₀ values ranging from 1.40?±?0.10 to 28.7?±?0.36?µM. Compound 3k displayed most potent cytotoxicity against lung cancer (NCI-H460) cells with an IC₅₀ value of 1.40?±?0.10?µM. 3k also arrested the G2/M phase of the cell cycle and induced distinctive apoptotic features on lung cancer cells. The apoptosis induction is supported by various cellular assays such as AO/EB, DAPI, and DCFDA staining studies including clonogenic assay. Extent of apoptosis was also analyzed by Annexin binding and JC-1 staining. Moreover, this method is amenable for the generation of a library of new class of stable bioactive enamino-2-oxindoles.     

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.     

The permeation of organic cations through cAMP-gated channels in mammalian olfactory receptor neurons

The permeation of monovalent organic cations through adenosine 3,5-cyclic monophosphate-(cAMP) activated channels was studied by recording macroscopic currents in excised inside-out membrane patches from the dendritic knobs of isolated mammalian olfactory receptor neurons (ORNs). Current-voltage relations were measured when bathing solution Na⁺ was replaced by monovalent organic cations. Permeability ratios relative to Na⁺ ions were calculated from changes in reversal potentials. Some of the small organic cations tested included ammonium (NH ₄ ⁺ ), hydroxylammonium and formamidinium, with relative permeability ratios of 1.41, 2.3 and 1.01 respectively. The larger methylated and ethylated ammonium ions studied included: DMA (dimethylammonium), TMA (tetramethylammonium) and TEA (tetraethylammonium) and they all had permeability ratios larger than 0.09. Even large cations such as choline, arginine and tris(hydroxymethyl)aminomethane (Tris) were appreciably permeant through the cAMP-activated channel with permeability ratios ranging from 0.19 to 0.7. The size of the permeating cations, as assessed by molecular weight, was a good predictor of the permeability. The permeability sequence of the cAMP-activated channel in our study was P_NH4 > P_Na > p_DMA > p_TMA > P_Choline > P_TEA. Higher permeability ratios of hydroxylammonium, arginine and tris(hydroxymethyl)aminomethane cannot be explained by ionic size alone. Our results indicate that: (i) cAMP-activated channels poorly select between monovalent cations; (ii) the pore dimension must be at least 6.5 × 6.5 Å, in order to allow TEA and Tris to permeate and (iii) molecular sieving must be an important mechanism for the permeation of large organic ions through the channels with specific ion binding playing a smaller role than in other structurally similar channels. In addition, the results clearly indicate that cyclic nucleotide-gated (CNG) channels in different cells are not the same, the olfactory CNG channel being different from that of the photoreceptors, particularly with respect to the permeation of large organic cations, which the ORN channels allow to permeate readily.This work was supported by the Australian Research Council of Australia.     

Modulation of glutathione level during butyrate-induced differentiation in human colon derived HT-29 cells

Glutathione plays an important role in various cellular functions including cell growth and differentiation. In the present study, cell differentiation was induced by butyrate in human colon cell line HT-29 and cellular thiol status was assessed. It was observed that butyrate-induced differentiation was associated with decrease in cellular GSH level and this was prominent at early stages of differentiation. Buthionine sulfoximine (BSO), a specific cellular GSH depleting agent, did not induce differentiation in cells but potentiated the differentiation induced by butyrate. Both BSO and butyrate individually and together inhibited cell growth. These studies suggest that cellular GSH level is modulated in butyrate-induced differentiation and decrease of GSH at the initial stage might facilitate cellular differentiation.