技术资料

Helicobacter pylori causes cellular vacuolation in host cells,a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT),a constitutively expressed secretory enzyme of H. pylori,in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium,thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay,we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (Ptextless0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably,vacuolation induced by WT was significantly reduced in the absence of GGT substrate,glutamine (Ptextless0.05) or in the presence of a competitive GGT inhibitor,serine-borate complex. Furthermore,the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT),although rGGT itself did not induce vacuolation independently. Similarly,the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally,we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively,our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases. View Publication

BACKGROUND: We previously identified a novel serine protease inhibitor (serpin),megsin,which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion,suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases. METHODS: We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases. RESULTS: Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml,which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18),55.6% were urinary megsin-positive,while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive,respectively. Among patients with chronic renal failure due to unknown causes (n = 74),18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073),12.3% were urinary megsin-positive,while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note,when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate,urinary megsin excretion increased as the stage progressed up to stage 3A,suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage,probably reflecting development of glomerulosclerosis. CONCLUSION: We established a high-sensitive megsin ELISA,which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases. View Publication

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here,we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains,both lacking canonical export signals,are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs-named 'cyclonals'-effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. View Publication

In Treponema denticola,a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola,and double- crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects,including highly condensed chromosomal DNA,compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover,chains of cells are formed by the cytoplasmic filament- deficient mutant,and those cells show reduced spreading in agarose,which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure,segregation,or the cell division process in Treponema. View Publication

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites,K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to textgreater 220 kDa,whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses,depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated,intact,permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting,indicating that these mAbs have potential for use in developing a field-based diagnostic test. View Publication