Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE

We purified and characterized a serine proteinase secreted by Acanthamoeba healyi to evaluate it as a possible virulence factor in the pathogenesis of granulomatous amoebic encephalitis (GAE). Ammonium sulfate precipitated culture supernatant of A. healyi OC-3A strain was purified by chromatography on CM-Sepharose, Sephacryl-S200, and Q-2 anion-exchange columns. The purified 33-kDa enzyme had a pH optimum of 8.0 and a temperature optimum of 40 C. Phenylmethylsulfonylfluoride and diisopropyl fluorophosphate, serine proteinase inhibitors, diminished activity of the enzyme to near zero. In addition to types I and IV collagen and fibronectin, the main components of the extracellular matrix, other proteins such as fibrinogen, IgG, IgA, albumin, and hemoglobin were also degraded by the enzyme. The broad substrate specificity of this secreted serine proteinase suggests that it may play an important role in pathogenesis of GAE by A. healyi.     

The Gossypium hirsutum TIR-NBS-LRR gene GhDSC1 mediates resistance against Verticillium wilt

Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome-wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR-NBS-LRR receptor-like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA-signalling-related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P-loop motif encoded by GhDSC1. This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana, and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.     

Three flavonoids targeting the beta-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori: crystal structure characterization with enzymatic inhibition assay

Flavonoids are the major functional components of many herbal and insect preparations and demonstrate varied pharmacological functions including antibacterial activity. Here by enzymatic assay and crystal structure analysis, we studied the inhibition of three flavonoids (quercetin, apigenin, and (S)-sakuranetin) against the beta-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori (HpFabZ). These three flavonoids are all competitive inhibitors against HpFabZ by either binding to the entrance of substrate tunnel B (binding model A) or plugging into the tunnel C near the catalytic residues (binding model B) mainly by hydrophobic interaction and hydrogen-bond pattern. Surrounded by hydrophobic residues of HpFabZ at both positions of models A and B, the methoxy group at C-7 of (S)-sakuranetin seems to play an important role for the inhibitor's binding to HpFabZ, partly responsible for the higher inhibitory activity of (S)-sakuranetin than those of quercetin and apigenin against HpFabZ (IC(50) in microM: (S)-sakuranetin, 2.0 +/- 0.1; quercetin: 39.3 +/- 2.7; apigenin, 11.0 +/- 2.5). Our work is expected to supply useful information for understanding the potential antibacterial mechanism of flavonoids.     

Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells

Notch signaling is critical for the stemness of radial glial cells (RGCs) during embryonic neurogenesis. Although Notch-signal-receiving events in RGCs have been well characterized, the signal-sending mechanism by the adjacent cells is poorly understood. Here, we report that conditional inactivation of mind bomb-1 (mib1), an essential component for Notch ligand endocytosis, in mice using the nestin and hGFAP promoters resulted in complete loss of Notch activation, which leads to depletion of RGCs, and premature differentiation into intermediate progenitors (IPs) and finally neurons, which were reverted by the introduction of active Notch1. Interestingly, Mib1 expression is restricted in the migrating IPs and newborn neurons, but not in RGCs. Moreover, sorted Mib1+ IPs and neurons can send the Notch signal to neighboring cells. Our results reveal that not only newborn neurons but also IPs are essential Notch-ligand-presenting cells for maintaining RGC stemness during both symmetric and asymmetric divisions.     

Characterization of the G‐quadruplex structure of a catalytic DNA with peroxidase activity

It has been reported that the complexes formed by hemin and some G‐quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. This kind of DNAzyme has received a great deal of attention. But to date, the actual G‐quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Herein, the G‐quadruplex structure of CatG4, a 21‐nucleotide DNA oligomer which was previously reported to bind hemin and the resulting complex exhibiting enhanced peroxidase activity, was characterized by fluorescence and circular dichroism measurements. The results suggest that the catalytically active form of CatG4 may be a unimolecular parallel quadruplex rather than a unimolecular chair‐type antiparallel quadruplex or a multistranded parallel quadruplex. In addition, the fluorescence analysis of labeled oligonucleotides may be developed as a supplementary tool for the study of DNA conformations. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 331–339, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Dietary supplementation with Chinese herbal powder enhances ileal digestibilities and serum concentrations of amino acids in young pigs

This study was designed to determine the effect of ultra-fine Chinese herbal powder as a dietary additive on serum concentrations and apparent ileal digestibilities (AID) of amino acids (AA) in young pigs. In Experiment 1, 60 Duroc x Landrace x Yorkshire piglets weaned at 21 days of age were randomly assigned to one of three treatments, representing supplementation with 0 or 2 g/kg of the powder, or 0.2 g/kg of colistin (an antibiotic) to corn- and soybean meal-based diets (n = 20 per group). Blood samples from five piglets per group were collected on days 7, 14, and 28 to determine serum AA concentrations. In Experiment 2, 12 barrows with an average initial body weight of 7.64 kg were randomly assigned to one of the three dietary treatments, followed by surgical placement of a simple T-cannula at the terminal ileum. All of the diets contained 0.1% titanium oxide as a digestibility marker. The samples of terminal ileal digesta were collected on day 7 for determining AID of AA. Results show that dietary supplementation with the herbal powder increased (P < 0.05) serum concentrations and AID of most AA by 10-50% and 10-16%, respectively. As an indicator of improved intestinal function, AID values of calcium were also enhanced in piglets supplemented with the herbal powder. Dietary supplementation of colistin increased serum concentrations and AID values of some AA by 8-44% and 10-15%, respectively, in comparison with the non-supplemented group. These novel findings demonstrate that the herbal powder can enhance the digestibility of dietary protein and the intestinal absorption of AA into the systemic circulation in post-weaning pigs, therefore providing a new mechanism for its growth- and immunity-promoting efficacy.