Inhibition of matrix metalloproteinase MMP-2 activates chloride current in human airway epithelial cells.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.     

分离自冬虫夏草可培养真菌的多样性研究

冬虫夏草是生长于青藏高原的一种名贵中药材。天然冬虫夏草及其微环境中生活着多种真菌。作者使用常规分离培养方法对冬虫夏草的真菌区系进行研究。从天然冬虫夏草的子座、菌核和菌膜3个部位共分离到572个真菌菌株,并根据形态特征将大部分菌株鉴定到37个不同的属。这些菌株经SSCP(single-strand conformation polymorphism)分析后,再根据nrDNAITS序列的相似性(以97%为阈值)共区分出92种不同的分类单元(operational taxonomic unit,OTU)。其中,属于子囊菌的菌株数及OTU数均比接合菌和担子菌多。从菌膜分离的菌株数及OTU数都明显多于子座和菌核。分离自子座的优势真菌是产黄青霉Penicillium chrysogenum,而分离自菌核和菌膜的优势真菌均为玫红假裸囊菌Pseudogymnoascus roseus。尚未最终鉴定的部分真菌可能为新的真菌物种。     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的抑制作用

目的观察乳酸杆菌DM9811发酵滤液及其主要成分对宫颈癌细胞株Hela细胞的体外增殖的影响,探索乳酸杆菌发酵滤液对宫颈癌细胞是否有抑制作用及解析作用的有效成分。方法用MTr法研究不同浓度乳酸杆菌DM9811发酵滤液在不同时间对Hela细胞的抑制作用,在此基础上研究脂肪酸、菌体核酸在不同时间对Hela细胞的抑制作用。结果不同浓度乳酸杆菌DM9811发酵滤液及相关物质在不同时间对Hela细胞的抑制作用显示：（1）乳酸杆菌DM9811发酵滤液各浓度组对Hela细胞的生长均有抑制作用,且这种抑制作用呈剂量-时间依赖方式。24、48、72h达到半数抑制率的发酵滤液浓度分别为8．9％、5．3％、3．8％。（2）乳酸杆菌DM9811发酵滤液脂肪酸对Hela细胞的生长有一定抑制作用,抑制率在7．0％～34．0％。（3）乳酸杆菌DM9811菌体核酸对Hela细胞的生长有抑制作用,抑制率为9．7％-53．4％,呈剂量一时间依赖方式。72h达到半数抑制率核酸的浓度为5．5μg/ml。结论乳酸杆菌DM9811发酵滤液对Hela细胞的生长具有显著的抑制作用,其中脂肪酸组分是有效成分之一。     

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

A genetic off-target event in a site-specific integration cell line expressing monoclonal antibody has no impact on commercial suitability

Site-specific integration (SSI) cell line systems are gaining popularity for biotherapeutic development and production. Despite the proven advantages for these expression hosts, the SSI system is still susceptible to rare off-target events and potential vector rearrangements. Here we describe the development process of an SSI cell line for production of an IgG1 monoclonal antibody (mAb-086). During cell line generational studies to assess suitability of clone C10 for commercial purposes, restriction fragment lengths of genomic DNA harboring the light chain (LC) were not in agreement with the predicted size. We first confirmed that the SSI landing-pad achieved occupancy of the desired expression plasmid. Additional investigation revealed that random integration had occurred, resulting in the acquisition of a partial copy of the LC and a full-length copy of the heavy chain (HC) at a different locus in the host genome. This off-target event had no impact on the genotypic consistency and phenotypic stability of the cell line, the production process, or the drug substance product quality. Given the genetic, phenotypic, and process consistency of the cell line, clone C10 was deemed suitable as a manufacturing cell line.     

Biochemical properties and expression profile of human prolyl dipeptidase DPP9

Dipetidyl peptidase 9 (DPP9) is a prolyl dipeptidase preferentially cleaving the peptide bond after the penultimate proline residue. The biological function of DPP9 is unknown. In this study, we have significantly improved the yield using Strep·Tactin^® purification system and characterized the biochemical property of DPP9. Moreover, the dimer interaction mode was investigated by introducing a mutation (F842A) at the dimer interface, which abolished the enzymatic activity without disrupting its quaternary structure. Furthermore, DPP9 was found ubiquitously expressed in fibroblasts, epithelial, and blood cells. Surprisingly, contrary to previous report, we found that the expression levels of DPP8 and DPP9 did not change upon the activation of the PBMC or Jurkat cells. These results indicate that the biochemical property of DPP9 is very similar to that of DPP8, its homologous protease. DPP9 and DPP8 are likely redundant proteins carrying out overlapping functions in vivo.