Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

NEG2, a short C-terminal segment (817–838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.     

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (k(cat)/K(m)) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.     

ModuleSearch: finding functional modules in a protein-protein interaction network

Many biological processes are performed by a group of proteins rather than by individual proteins. Proteins involved in the same biological process often form a densely connected sub-graph in a protein-protein interaction network. Therefore, finding a dense sub-graph provides useful information to predict the function or protein complex of uncharacterised proteins in the sub-graph. We developed a heuristic algorithm that finds functional modules in a protein-protein interaction network and visualises the modules. The algorithm has been implemented in a platform-independent, standalone program called ModuleSearch. In an interaction network of yeast proteins, ModuleSearch found 366 overlapping modules. Of the modules, 71% have a function shared by more than half the proteins in the module and 58% have a function shared by all proteins in the module. Comparison of ModuleSearch with other programs shows that ModuleSearch finds more sub-graphs than most other programs, yet a higher proportion of the sub-graphs correspond to known functional modules. ModuleSearch and sample data are freely available to academics at http://bclab.inha.ac.kr/ModuleSearch.     

巨噬细胞与树突状细胞在正常成人真皮内分布形式的研究

目的观察正常成人真皮内巨噬细胞和树突状细胞的分布、形态学和密度。方法正常人8例,取面部、躯干、四肢近端、四肢远端、手掌和足跖6个部位皮肤,进行纵行与水平切片。CD1a、CD68、OKM5单克隆抗体和FⅩⅢa多克隆抗体染色。结果CD1a和FXⅢa阳性细胞多位于皮肤附属器和血管周围。CD68阳性细胞在真皮浅层呈网状分布,真皮深层密度明显降低。各个部位的OKM5阳性细胞数较少。真皮内各个部位CD68阳性细胞高于相同部位CD1a、FⅩⅢa和OKM5阳性细胞(P<0·0001)。结论在真皮内存在大量的呈网状分布的CD68阳性的单核-巨噬细胞;同时在真皮,特别是浅层,有较多的树突状细胞,如郎格汉斯细胞和真皮树突状细胞,但其数量远低于CD68阳性的单核-巨噬细胞。CD68阳性的单核-巨噬细胞和真皮内的树突状细胞,是真皮内的主要防御细胞。     

Leaf nitrogen dioxide uptake coupling apoplastic chemistry, carbon/sulfur assimilation, and plant nitrogen status

Emission and plant uptake of atmospheric nitrogen oxides (NO + NO₂) significantly influence regional climate change by regulating the oxidative chemistry of the lower atmosphere, species composition and the recycling of carbon and nutrients, etc. Plant uptake of nitrogen dioxide (NO₂) is concentration-dependent and species-specific, and covaries with environmental factors. An important factor determining NO₂ influx into leaves is the replenishment of the substomatal cavity. The apoplastic chemistry of the substomatal cavity plays crucial roles in NO₂ deposition rates and the tolerance to NO₂, involving the reactions between NO₂ and apoplastic antioxidants, NO₂-responsive germin-like proteins, apoplastic acidification, and nitrite-dependent NO synthesis, etc. Moreover, leaf apoplast is a favorable site for the colonization by microbes, which disturbs nitrogen metabolism of host plants. For most plant species, NO₂ assimilation in a leaf primarily depends on the nitrate (NO₃ ⁻) assimilation pathway. NO₂–N assimilation is coupled with carbon and sulfur (sulfate and SO₂) assimilation as indicated by the mutual needs for metabolic intermediates (or metabolites) and the NO₂-caused changes of key metabolic enzymes such as phosphoenolpyruvate carboxylase (PEPc) and adenosine 5′-phosphosulfate sulfotransferase, organic acids, and photorespiration. Moreover, arbuscular mycorrhizal (AM) colonization improves the tolerance of host plants to NO₂ by enhancing the efficiency of nutrient absorption and translocation and influencing foliar chemistry. Further progress is proposed to gain a better understanding of the coordination between NO₂–N, S and C assimilation, especially the investigation of metabolic checkpoints, and the effects of photorespiratory nitrogen cycle, diverse PEPc and the metabolites such as cysteine, O-acetylserine (OAS) and glutathione.     

Non-structural and nucleocapsid proteins of Punta Toro virus induce apoptosis of hepatocytes through both intrinsic and extrinsic pathways

Punta Toro virus (PTV; family Bunyaviridae , genus Phlebovirus ) causes severe hepatic damage through brisk apoptosis of hepatocytes. In the present study, two viral proteins encoded by the S segment of the viral genome, non-structural (NSs) and nucleocapsid protein (N), were examined for their roles in apoptosis. Expression of NSs in HepG2 cells led to apoptosis in 45% of transfected cells, and with N, 28%, on average. These levels represent a four- to an eightfold increase over cells transfected with the mutated protein vectors. Caspase-3, -8 and -9 activities were increased by N protein when compared with the control NC ( P < 0.05), and by NSsA and NSsB, as compared to control NSsC ( P < 0.01). Treatment of the transfected cells with caspase-8 or -9 inhibitors markedly decreased apoptosis. Neutralization of TNF-α or Fas ligand had no effect on apoptosis. These results indicate that both NSs and N are responsible for causing hepatocyte apoptosis by triggering the extrinsic caspase-8 and intrinsic caspase-9 pathways.     

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of moderately halophilic bacteria isolated from the Weihai Solar Saltern (China)

A total of 45 moderately halophilic bacteria was isolated from sediment and saline water collected from the Weihai Solar Saltern (China). The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The halophilic strains were tested for their antimicrobial activity. Cytotoxicity assay was performed to determine which of the halophilic strains could inhibit proliferation of human hepatocellular carcinoma Bel 7402 cells. Our results showed that all of the isolated 45 strains displayed moderately halophilic characteristics. Phylogenetic analysis indicated that 17 of the isolated strains were related to the phylum Firmicutes and belonged to four genera, Bacillus, Halobacillus, Planococcus and Salinicoccus. The other strains identified as genus of Halomonas belonged to phylum γ-Proteobacteria. Most of the halophilic bacterial strains showed potent activities against Gram-positive bacteria, human pathogenic fungi and plant pathogenic fungi. In addition, the crude extracts from 14 halophilic bacterial strains showed cytotoxic activity against tumor cells Bel 7402, and five of them showed remarkable activities with IC₅₀ less than 40 μg ml⁻¹. Our results suggest that the moderately halophilic bacteria may be developed as promising sources for the discovery of novel bioactive substances.     

A new species of <Emphasis Type="Italic">Zygophiala</Emphasis> associated with the flyspeck complex on apple from China

Zygophiala qianensis is described as a new fungal species associated with the cuticle of apple fruit sampled from an orchard in Shaanxi Province, China. Conidiophores were separate, arising from superficial hyphae, erect, scattered, subcylindrical, irregularly flexuous, consisting of four parts: a hyaline supporting cell that gives rise to a smooth, dark brown stipe, terminating in a finely verruculose, medium brown apical cell that gives rise to (1–)2(–3) medium brown, finely verruculose, doliiform to ellipsoidal, polyblastic conidiogenous cells, with 1–2 prominent scars, apical and lateral, darkened, thickened. Conidia were solitary, fusiform to obclavate, hyaline, smooth and thick-walled, transversely (0–)1(–7)-septate, mostly 1–2-septate, prominently constricted at the septum; apex obtuse, base subtruncate, with a darkened, thickened hilum. Zygophiala qianensis is compared morphologically to other species of Zygophiala, and a phylogenetic analysis of their DNA sequence data is presented.     

Synthesis and conformational analysis of a locked analogue of carbovir built on a bicyclo[3.1.0]hex-2-enyl template

The synthesis and biological evaluation of a carbovir analogue (5) built on a bicyclo[3.1.0]hex-2-enyl template is described. A conformational analysis using density functional theory at the B3LYP/6-31G* level has been carried out on the rigid pseudosugar template of 5, the cyclopentene moiety of carbovir and the bicyclo[3.1.0]hex-2-yl pseudosugars of two isomeric carbonucleosides (12 and 13) containing exo- and endo-fused cyclopropane rings. The results show that while the planar configuration of the fused cyclopentane ring of compound 5 helps retain weak anti-HIV activity, the ability of the cyclopentene ring of carbovir to easily adopt a planar or puckered conformation with little energy penalty may prove to be a crucial advantage. The bicyclo[3.1.0]hex-2-yl nucleosides 12 and 13 that were inactive against HIV exhibited stiffer resistance to having a planar, fused cyclopentane moiety.