Effects of non-ionic surfactants on the interactions between cellulases and tannic acid: a model system for cellulase-poly-phenol interactions

Addition of non-ionic surfactants (NIS) is known to accelerate enzymatic lignocellulose hydrolysis. The mechanism behind this accelerating effect is still not elucidated but has been hypothesized to originate from favorable NIS-lignin interactions which alleviate non-productive adsorption of cellulases to lignin. In the current work we address this hypothesis using tannic acid (TAN) as a general poly-phenolic model compound (for lignin and soluble phenolics) and measure the mutual interactions of cellulases (CBHI, CBHII, EGI, EGII and BG), TAN and NIS (Triton X-100) using isothermal titration calorimetry (ITC). The experimental results suggest rather strong enzyme-specific interactions with TAN in reasonable agreement with enzyme specific lignin inhibition found in the literature. Enzyme-TAN interactions were disrupted by the presence of NIS by a mechanism of strong TAN-NIS interaction. The presence of NIS also alleviated the inhibitory effect of TAN on cellulase activity. All together the current work provides strong indications that favorable NIS-poly-phenol interactions alleviate non-productive cellulase-poly-phenol interactions and hence may provide a mechanism for the accelerating effect of NIS on lignocellulose hydrolysis.     

Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1.

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol.     

Regulation of murine airway surface liquid volume by CFTR and Ca2+-activated Cl- conductances

Two Cl(-) conductances have been described in the apical membrane of both human and murine proximal airway epithelia that are thought to play predominant roles in airway hydration: (1) CFTR, which is cAMP regulated and (2) the Ca(2+)-activated Cl(-) conductance (CaCC) whose molecular identity is uncertain. In addition to second messenger regulation, cross talk between these two channels may also exist and, whereas CFTR is absent or defective in cystic fibrosis (CF) airways, CaCC is preserved, and may even be up-regulated. Increased CaCC activity in CF airways is controversial. Hence, we have investigated the effects of CFTR on CaCC activity and have also assessed the relative contributions of these two conductances to airway surface liquid (ASL) height (volume) in murine tracheal epithelia. We find that CaCC is up-regulated in intact murine CF tracheal epithelia, which leads to an increase in UTP-mediated Cl(-)/volume secretion. This up-regulation is dependent on cell polarity and is lost in nonpolarized epithelia. We find no role for an increased electrical driving force in CaCC up-regulation but do find an increased Ca(2+) signal in response to mucosal nucleotides that may contribute to the increased Cl(-)/volume secretion seen in intact epithelia. CFTR plays a critical role in maintaining ASL height under basal conditions and accordingly, ASL height is reduced in CF epithelia. In contrast, CaCC does not appear to significantly affect basal ASL height, but does appear to be important in regulating ASL height in response to released agonists (e.g., mucosal nucleotides). We conclude that both CaCC and the Ca(2+) signal are increased in CF airway epithelia, and that they contribute to acute but not basal regulation of ASL height.     

Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences – including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps – are still limited by the lack of high‐quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype‐resolved genome resequencing at population scale, we investigated properties of linked‐read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high‐coverage data (46–68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25–4.6 Mb (N50) and 0.27–0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher‐quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase‐sized genomes like birds, linked‐read sequencing at moderate depth opens an affordable avenue towards haplotype‐resolved genome resequencing at population scale.     

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.     

Chemical Shifts Provide Fold Populations and Register of β Hairpins and β Sheets

A detailed analysis of peptide backbone amide (H_N) and Hα chemical shifts reveals a consistent pattern for β hairpins and three-stranded β sheets. The Hα’s at non-hydrogen-bonded strand positions are inwardly directed and shifted downfield ~1 ppm due largely to an anisotropy contribution from the cross-strand amide function. The secondary structure associated Hα shift deviations for the H-bonded strand positions are also positive but much smaller (0.1–0.3 ppm) and the turn residues display negative Hα chemical shift deviations (CSDs). The pattern of (H_N) shift deviations is an even better indicator of both hairpin formation and register, with the cross-strand H-bonded sites shifted downfield (also by ~1 ppm) and with diagnostic values for the first turn residue and the first strand position following the turn. These empirical observations, initially made for [2:2]/[2:4]-type-I' and -II' hairpins, are rationalized and can be extended to the analysis of other turns, hairpin classes ([3:5], [4:4]/[4:6]), and three-stranded peptide β-sheet models. The Hα’s at non-hydrogen-bonded sites and (H_N)’s in the intervening H-bonded sites provide the largest and most dependable measures of hairpin structuring and can be used for melting studies; however the intrinsic temperature dependence of (H_N) shifts deviations needs to reflect the extent of solvent sequestration in the folded state. Several observations made in the course of this study provide insights into β-sheet folding mechanisms: (1) The magnitude of the (H_N) shifts suggests that the cross-strand H-bonds in peptide hairpins are as short as those in protein β sheets. (2) Even L-Pro-Gly turns, which are frequently used in unfolded controls for hairpin peptides, can support hairpin populations in aqueous fluoroalcohol media. (3) The good correlation between hairpin population estimates from cross-strand H-bonded (H_N) shift deviations, Hα shift deviations, and structuring shifts at the turn locus implies that hairpin folding transitions approximate two-state behavior. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants

The functional role of endostatin's affinity for heparan sulfates was addressed using an ex vivo bone angiogenesis model. Capillary-like sprouts showed prominent expression of collagen XVIII/endostatin. Outgrowth of endothelial cells was not altered in the absence of collagen XVIII but inhibited by the addition of recombinant endostatin. Mutant non-heparan sulfate binding endostatin and the collagen XV endostatin homologue were ineffective. The ability of mutant endostatin to bind to capillary structures was reduced when compared to endostatin. Endostatin-XV completely failed to bind to endothelial cells. Our data indicate that endostatin's angiostatic function is heparan sulfate-dependent, and that in situ-binding of endostatin to endothelial cells is increased by heparan sulfates.