FGFR3 contributes to intestinal crypt cell growth arrest

Fibroblast growth factors (FGFs) are important regulators of the dynamic development and turnover of tissues. Among FGF receptors, FGFR3 expression is confined in the intestinal crypts. We examined FGFR3-deficient mice and saw increased intestinal crypt depth but no change in villae length or in the distribution of differentiated intestinal cells, suggesting that the impact of lack of FGFR3 was limited to the progenitor cell compartment. Accordingly, enhancement of intestinal crypt proliferation was observed in FGFR3 mutant mice and interestingly, upon anti-FGFR3 antibody administration in wild type mice. Moreover, injection of FGF18, a ligand of FGFR3, in wild type mice resulted in decreased cell proliferation within the intestinal crypts. In addition, we found that ERK level of activation was increased in FGFR3-deficient intestinal epithelium. In vitro studies showed that ERK, AKT and activation was regulated by FGFs and that ERK level of activation was inversely correlated to FGFR3 level of expression in the intestinal crypt cells. Furthermore, effects of FGF18 on ERK and AKT activation paralleled FGFR3 effects on these intracellular targets. Our data indicate that FGF18 and FGFR3 are involved, possibly as partners, in the control of intestinal precursor cell proliferation.     

In vitro-refolding of a single-chain Fv fragment in the presence of heteroaromatic thiols

The aim of the present work was to explore the use of heteroaromatic thiol compounds, namely derivatives of pyridine and pyrimidine, as redox reagents for the in vitro-refolding of a recombinantly expressed single-chain Fv fragment (scFvOx). The mixed disulfide of scFvOx with glutathione was used as a starting material, while reduced glutathione, 4-mercaptopyridine, 2-mercaptopyrimidine, 2-mercaptopyridine N-oxide, and the mercaptobenzene derivative thiosalicylic acid, respectively, served as catalysts for the formation of native disulfide bonds during renaturation. In contrast to thiosalicylic acid, and despite their significantly lower thiol pKa values, none of the heteroaromatic thiol compounds accelerated the apparent kinetics of in vitro-refolding compared to the naturally occurring peptide glutathione. However, significantly improved renaturation yields were observed in the presence of 4-mercaptopyridine and 2-mercaptopyrimidine, demonstrating the usefulness of aromatic thiol compounds as reagents for the in vitro-refolding of antibody fragments.     

The white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>: conditions for the production of lignin-degrading enzymes

Investigating optimal conditions for lignin-degrading peroxidases production by Phanerochaete chrysosporium (P. chrysosporium) has been a topic for numerous researches. The capability of P. chrysosporium for producing lignin peroxidases (LiPs) and manganese peroxidases (MnPs) makes it a model organism of lignin-degrading enzymes production. Focusing on compiling and identifying the factors that affect LiP and MnP production by P. chrysosporium, this critical review summarized the main findings of about 200 related research articles. The major difficulty in using this organism for enzyme production is the instability of its productivity. This is largely due to the poor understanding of the regulatory mechanisms of P. chrysosporium responding to different nutrient sources in the culture medium, such as metal elements, detergents, lignin materials, etc. In addition to presenting the major conclusions and gaps of the current knowledge on lignin-degrading peroxidases production by P. chrysosporium, this review has also suggested further work, such as correlating the overexpression of the intra and extracellular proteins to the nutrients and other culture conditions to discover the regulatory cascade in the lignin-degrading peroxidases production process, which may contribute to the creation of improved P. chrysosporium strains leading to stable enzyme production.     

Mechanistic insights into replication termination as revealed by investigations of the Reb1-Ter3 complex of Schizosaccharomyces pombe

Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the “sweepase” Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.     

Chitinase production by Beauveria felinaRD 101: optimization of parameters under solid substrate fermentation conditions

Major parameters affecting the production of chitinase by Beauveria felinaRD 101 under solid substrate fermentation conditions have been optimized. Wheat bran moistened with 100 MS-HCl medium adjusted to pH 5.0, inoculated with 1 × 10¹⁰ conidia g^–1 initial dry bran and incubated at 28 °C for 6 days produced maximum chitinase activity of 6.34 U g^–1 initial dry substrate.     

Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.     

Bioconjugated quantum rods as targeted probes for efficient transmigration across an in vitro blood-brain barrier

We report here, what we believe to be the first time, the successful transport of bioconjugated quantum rods (QRs) across an in vitro blood-brain barrier (BBB) model via a receptor-mediated transport, as well as the use of QR multiplexing technique to compare simultaneously the transmigration efficiency of different biomolecules across the BBB. The migration rate of bioconjugated QRs crossing the in vitro BBB was found to be concentration- and time-dependent. This work illustrates a nanoparticle-based platform that will not only allow a direct visualization of the transmigration ability of various kinds of biomolecules across the BBB, but also facilitate the development of novel diagnostic and therapeutic nanoprobes for early diagnosis and therapy of various disorders of the brain following systemic administration.     

Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco.

Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B. thuringiensis corn, rice, and potato but not from B. thuringiensis canola, cotton, and tobacco. Nonsterile soil and sterile hydroponic solution in which B. thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B. thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively. No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B. thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-B. thuringiensis plant counterparts or no plants, had been grown. All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize. The reasons for the differences between species in the exudation from roots of the toxins are not known. The released toxins persisted in soil as the result of their binding on surface-active particles (e.g. clay minerals, humic substances), which reduced their biodegradation. The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects.