10mM EDTA Research Articles

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

Purification of Bacteriocin from Lactobacillus plantarum KY449289 and its Role in the Preservation of Mixed Fruit Juice

Background:: The aim of this study is to isolate, extract and purify the antibacterial protein from Lactobacillus plantarum KY449289 and to assess its bio preservative potential. Objective:: The present study was conducted to evaluate the laxative activity of Hydroethanolic Pericarp Extract of sapindus emarginatus (HESE) in animal models. Methods:: Bacteriocins are antimicrobial peptides synthesized ribosomally which can be used as bio-preservative reducing the risk of chemical preservative’s effect and also replacing the thermal treatments. This study aims to isolate, purify the antibacterial protein from Lactobacillus plantarum KY449289 and to assess its bio preservative potential. In this study, twenty strains were isolated from a yogurt sample and preliminary characterization was carried out, such as morphological, biochemical, and molecular levels as a preliminary assay. Results:: The characterized strains were assessed for the antibacterial activity by well diffusion assay, whereas all twenty strains were shown to have a antibacterial activity against foodborne pathogens, among which two strains VITMM04 and VITMM05 shows the highest zone of inhibition of 15 mm and 10 mm respectively against Listeria monocytogenes. The potent isolate VITMM05 solely showed a broad spectrum antibacterial activity against the pathogens. These strains were further subjected to optimization, purification, and application studies. A high increase in the inhibition zone was recorded with an optimum temperature of 35°C and its activity was found to be stable up to 85°C, optimum pH 6, and 5 mM of EDTA as an enhancer surfactant. On subjected to RP-HPLC, the purified sample showed a distinct peak at 2.192 min corresponding to the peak at 2.192 min for standard bacteriocin. Conclusion:: The six-fold purified bacteriocin was effective in controlling 87% of the microbial population with 20mm zone of inhibiton against the Listeria monocytogenes and this shows the effective role of purified bacteriocin in inhibiting bacterial growth in a greater extent.

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage, EDTA and nisin

P22 phage >105 PFU ml−1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25 mM and concentrations of nisin >1,200 µg ml−1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (102 PFU ml−1), and low EDTA (1.25 mM) and nisin (9.375 µg ml−1) concentrations. A reduction of 70% in the mature biofilm was possible when 107 PFU ml−1 of P22 phage, 20 mM of EDTA and 150 μg ml−1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.

Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic) using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i) for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii) for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii) for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua) and green tea (Camellia sinensis) was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

The mechanism for keratinocyte detaching from pH-responsive chitosan

In this study, we compared the detachment ratio of HaCaT and Hs68 cells from pH-responsive chitosan surface by raising medium pH from 7.20 to 7.65 for 60 min. The detachment ratio of elongated Hs68 cells was over 75%, but that of round-shaped HaCaT cells was less than 50%, even extending the incubation time to 6 h or enhancing the cytoskeletal contractile force with the Rho activator CN01. However, the addition of 2 mm of EDTA into the medium at pH 7.65 could effectively detach HaCaT cells (detachment ratio > 90%), indicating that the calcium ion played an important role in the detachment process. Therefore, the family of Ca+2-dependent integrin receptors was examined by RT-PCR, real-time PCR and immunocytochemistry. It was found the expression of integrin β4 (ITGb4) was HaCaT cell-specific and the mRNA level of ITGb4 in undetached HaCaT cells was significantly higher than that in detached ones. By modulating ITGb4 activity with specific functional blocking antibody ASC-8, the detachment ratio of HaCaT cells could be increased to be greater than 85%. Conversely, the addition of the ligand of ITGb4 laminin into the culture system decreased the medium pH-induced detachment ratio for HaCaT cells, but not for Hs68 cells. Further addition of ASC-8 could rescue the effect of laminin on preventing the detachment of HaCaT cells from pH-sensitive chitosan surface. Therefore, this study demonstrated the interaction of ITGb4 and laminin played an important role in controlling the detachment of HaCaT cells on pH-responsive chitosan.

Halotolerant, acid‐alkali stable, chelator resistant and raw starch digesting α‐amylase from a marine bacterium Bacillus subtilis S8–18

A halotolerant α-amylase having the ability of digesting the insoluble raw starches was characterized from Bacillus subtilis S8-18, a marine sediment isolate from Palk Bay region. The electrophoresis techniques unveiled that the α-amylase was indeed a monomer with a molecular weight of 57 kDa. The optimum temperature and pH for the enzyme activity were 60 °C and 6.0 respectively. The enzyme was highly stable for 24 h over a wide range of pH from 4.0 to 12.0 by showing 84-94% activity. Interestingly, by retaining 72% activity even after 24 h, the enzyme also showed tolerance towards 28% NaCl. The α-amylase retained a minimum of 93% residual activity in 1 mM concentration for the selected divalent metal ions. The enzyme was found to be chelator resistant as it remained unaffected by 1 mM of EDTA and exhibited 96% activity even at 5 mM concentration. Furthermore, though 1% SDS caused remarkable reduction (68%) in amylase activity, the enzyme showed tolerance towards other detergents (1% of Triton-X and Tween 80) with 85% activity. Additionally, the α-amylase enzyme is capable of hydrolyzing the insoluble raw starch substrates which was evident from the scanning electron microscopic (SEM) and spectrophotometric analyses.

시설재배지에서 킬레이트제 처리가 양분 유효도와 배추생육에 미치는 영향

시설재배지 토양의 축적양분인 염류를 활용하기 위해 EC가 높은 토양에 여러 킬레이트제를 처리하여 토양의 화학성과 작물의 무기성분 흡수량을 비교 분석하였다. DTPA와 EDTA를 처리한 구에서 킬레이트제는 Ca, Mg, K, P등의 다량원소 뿐만 아니라 Fe, Cu, Mn, Zn 등의 미량원소와 킬레이트 하는 능력이 뛰어났다. 그러나, 토양의 ECDTPA를 처리한 는 EDTA를 처리한 토양보다 낮아졌다. 킬레이트제가 처리된 토양에서 생육한 배추의 무기성분 흡수량을 살펴본 결과 DTPA 0.5 mM 처리한 구에서 배추의 T-N, <TEX>$P_2O_5$</TEX>, <TEX>$K_2O$</TEX>, 그리고 Fe의 흡수량은 다른 처리구보다 높았다. 이로부터 시설재배지의 집적된 양분을 킬레이트화하여 작물의 양분 이용성을 증대하는 데 효과적인 킬레이트제는 DTPA이며, 적정한 농도는 0.5 mM로 나타났다. This study is conducted to evaluate the effects of chelating agents for improving plant growth and reusing accumulated nutrients in soils of plastic film house. Two experiments were carried out at follows: i) The incubation test was conducted using soils treated with 0, 300 mM of EDTA and DTPA to examine the availability of nutrients. ii) For the pot test, chinese cabbages were cultivated in soils with 0, 0.1, 0.5, 1, and 5 mM of EDTA and DTPA to examine the impacts of plant growth response. The application of chelating agents increased ther availability of soil nutrients in the following order: DTPA > EDTA > control. Inorganic concentration of chinese cabbages in DTPA treatments consderably increased in nitrogen, phosphate, iron and aluminium contents than that of the other treatments. The optimal concentration of DTPA for vigorous plant growth as 0.5 mM. Thus, DTPA was more effective than other chelating agents for healty growth of cabbages and the availability of nutrients accumulated in plastic film house.

Ischemic preconditioning activates caveolae-mediated eNOS signalling and elicits cardioprotection through S-nitrosylation of the subsarcolemmal mitochondria

BackgroundRecent studies suggest that caveolae transduce endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) signalling in ischemic preconditioning (IPC) hearts. And the NO-mediated protein S-nitrosylation (SNO) has been shown...

Controlled hydrothermal growth of ZnO nanostructures by sequestering the Zn metal ions with the chelating agent EDTA

In the present work, a controlled growth of ZnO nanostructures by manipulating Zn metal ion concentration by the chelating action of ethylene diaminetetra acetic acid in hydrothermal method is studied. EDTA produces metal–chelate complex by the formation of bidentate ligand with Zn 2+ in the solution and diminishes the reactivity of Zn metal cations. Concentration of EDTA in the mother solution was varied in different ranges like 3, 5 and 10 mM while retaining the zinc metal salt and the NaOH concentration the same. Three different morphologies of wurtzite structured ZnO nanostructures such as nanorods-bunch, separate/discrete uniformly sized hexagonal nanorods and tapered flower petals like shapes are achieved by 3, 5 and 10 mM strengths of EDTA, respectively. The medium concentration 5 mM of EDTA is found to have moderate control over producing ZnO nanostructures of uniform diameter and a high aspect (length to diameter) ratio. An array of vertically aligned free standing ZnO nanorods with uniform spacing is successfully achieved by the addition of 5 mM of EDTA in the mother solution and the same is studied for its fluorescence property at an excitation of 325 nm and it has exhibited a characteristic UV emission of ZnO around 383 nm.

Approaches to improve the stability of the antiviral agent UC781 in aqueous solutions

In this work, we evaluated the chemical stability profiles of UC781 based solutions to identify excipients that stabilize the microbicidal agent UC781. When different antioxidants were added to UC781 in sulfobutylether-β-cyclodextrin (SBE-β-CD) solutions and subjected to a 50 °C stability study, it was observed that EDTA was a better stabilizing agent than sodium metabisulfite, glutathione or ascorbic acid. Some antioxidants accelerated the degradation of UC781, suggesting metal-catalyzed degradation of UC781. Furthermore, we observed substantial degradation of UC781 when stored in 1% Tween 80 and 1% DMSO solutions alone or in those with 10 mM EDTA. On the other hand, improved stability of UC781 in the presence of 100 and 200 mM of EDTA was observed in these solutions. The addition of both EDTA and citric acid in the stock solutions resulted in recovery of more than 60% of UC781 after 12 weeks. Generally, 10% SBE-β-CD in the presence of EDTA and citric acid stabilized UC781 solutions: the amount of UC781 recovered approaching 95% after 12 weeks of storage at 40 °C. We also showed that the desulfuration reaction of the UC781 thioamide involves oxygen by running solution stability studies in deoxygenated media. Improved stability of UC781 in the present study indicates that the incorporation of EDTA, citric acid and SBE-β-CD and the removal of oxygen in formulations of this drug will aid in increasing the stability of UC781 where solutions of the drug are required.

Cloning and characterization of a xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL-001

The KRICT PX1 gene (GB: FJ380951) consisting of 996 bp encoding a protein of 332 amino acids (38.1 kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50 °C with K m value of 5.35 and 3.23, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH 4Cl, CaCl 2, MgCl 2, MnCl 2, and CsCl 2 at 1 mM, but affected by CuSO 4, ZnSO 4, and FeCl 3. One mM of EDTA, 2-mercaptoethanol, and PMSF did not affect the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. A similarity analysis of the amino acids in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_03040360), 70% identity with intracellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB: ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with glycosyl hydralase (GH) family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites, and this was confirmed through PROSITE scan. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.

Efficient Isolation of Mouse Liver NKT Cells by Perfusion

BackgroundNKT cell is a population of unconventional T cells that mediate both innate and adaptive T cell responses. Since NKT cells are most abundant in the liver, much of NKT biology has been learnt from studies of NKT cells isolated from liver. This is a cumbersome procedure with variations in cell yield.ResultsBased on recent evidence that NKT cells reside in liver vascular compartment, we developed a simple method to isolate NKT cells by perfusion with PBS-containing 10 mM of EDTA. The number and cell surface phenotype of liver NKT cells recovered by perfusion and by the traditional method were comparable. The yield of other lymphocytes was also comparable.Conclusion/SignificanceOur data demonstrated that liver lymphocytes can be efficiently isolated by simple perfusion. These data provide a convenient method to isolate liver lymphocyte while preserving liver tissue for other analysis.

A fast, simple, and reliable hydrophilic interaction liquid chromatography method for the determination of ascorbic and isoascorbic acids

A reliable method for the determination of total vitamin C must be able to resolve ascorbic acid (AA) and the epimeric isoascorbic acid (IAA) and determine the sum of AA and its oxidized form dehydroascorbic acid. AA and IAA are polar molecules with a low retention time in conventional reversed phase systems, and hence of difficult resolution. Hydrophilic interaction chromatography using a TSKgel Amide-80 stationary phase with isocratic elution was successful in resolving the two epimers. The column was compatible with injections of high concentrations of metaphosphoric acid, tris(2-carboxyethyl)-phosphine, and EDTA without drift of baseline and retention time. Total AA and IAA were extracted, stabilized, and reduced in one step at 40 °C, using 5% m-phosphoric acid, 2 mM of EDTA, and 2 mM of tris(2-carboxyethyl)-phosphine as reducing agent. This simple, fast, and robust hydrophilic interaction chromatography-DAD method was applied for the analysis of food products namely fruit juices, chestnut, and ham and also in pharmaceutical and multivitamin tablets. Method validation was performed on the food products, including parameters of precision, accuracy, linearity, limit of detection, and quantification (LOQ). The absence of matrix interferences was assessed by the standard addition method and Youden calibration. The method was fast, accurate, and precise with a LOQ(AA) of 1.5 mg/L and LOQ(IAA) of 3.7 mg/L. The simple experimental procedure, completed in 1 h, the possibility of using IAA as an internal standard, and low probability of artifacts are the major advantages of the proposed method for the routine determination of these compounds in a large number of samples.

ADAMTS-13 Has a Disulfide Bond Reduction Activity on Von Willebrand Factor

Von Willebrand factor (VWF) multimers tether platelets to subendothelium exposed at the site of vessel injury to initiate the bleeding arrest. Upon synthesized, VWF multimers are either constitutively secreted or packed into the storage granules, where they are enriched in ultra-large (UL) multimers that are active in forming spontaneous high strength bonds with the GP Ib-IX-V complex on platelets. This hyper-reactivity of ULVWF multimers is in contrast to VWF multimers circulating in plasma (pVWF) that need to be activated by modulators or high fluid shear stress to aggregate platelets. The biochemical and structural bases for the functional difference between ULVWF and pVWF multimers are not known. We have recently shown that a portion of pVWF, but not ULVWF multimers contain surface exposed free thiols in the D3 and C domains. High fluid shear stress promotes the formation of new disulfide bonds utilizing the thiols to enhance VWF binding to platelets, suggesting that the shear-induced thiol-disulfide exchange may serve as a mechanism for the shear-induced activation of pVWF multimers. ULVWF freshly secreted from endothelial cells forms string-like structures that can be elongated by pVWF multimers through a covalent means. The different thiol distribution between ULVWF and its plasma counterpart may be caused by the former being cleaved by the zinc metalloprotease ADAMTS-13 at a single peptide bond of Y1065-M1606 in the A2 domain. Here, we provide several lines of evidence to demonstrate that ADAMTS-13 also contains a reductase-like activity that plays a role in cleaving ULVWF strings under flow conditions and maintaining circulating VWF multimers in an inactive (thiol) state. First, more than 90% of pVWF non-specifically adhered to the surface of a cone-plate viscometer when pVWF was exposed to a pathological high shear stress of 100 dyn/cm2 for 3 min at 37°C. The adhesion was prevented by recombinant (r) ADAMTS-13 or a truncation mutant that lacked the catalytic domain. Second, rADAMTS-13 prevented the shear-induced thiol-disulfide exchange so that free thiols remained in pVWF after shear exposure. This activity was not blocked by 5 mM of EDTA and was detectable with the N-terminal truncated mutant, suggesting that it is independent of the VWF-cleaving activity. We further found that rADAMTS-13 was able to reduce disulfide bonds, converting the disulfide forms of sheared pVWF to the thiol forms, suggesting that ADAMTS-13 prevents the thiol-disulfide exchange by disulfide bond reduction, not a steric hindered effect. Third, ADAMTS-13 contains the surface exposed thiol(s) that is necessary for the metalloprotease to attack and break a disulfide bond. Unlike VWF multimers, these thiols remained after the metalloprotease was exposed to a pathological high shear stress of 100 dyn/cm2. Fourth, using a series of N- and C-terminal truncation mutants, we located the thiol(s) potentially involved in VWF reduction to the 2nd to 8th TSP-1 motifs and CUB-1 domain of ADAMTS-13. Finally, ethylmaleimide (NEM), which blocks free thiols, did not inhibit rADAMTS-13 to cleave pVWF multimers under static conditions and in the presence of urea and barium. NEM-treated rADAMTS-13 retained only 27.5±4.9% activity in cleaving ULVWF strings under flow conditions as compared to untreated enzyme. These data characterizes a novel mechanism that plays a regulatory role in cleaving ULVWF strings and maintaining the circulating pVWF multimers in inactive forms.

Shear Stress Induced Increase in the Forced (UL)VWF Domain Unfolding Is Non-Proteolytically Regulated by ADAMTS-13

Hemostasis is initiated by tethering platelets to the site of vessel injury, a process mediated by the interaction of the GP Ib-IX-V complex on platelets and von Willebrand Factor (VWF) in the subendothelial matrix. The adhesion activity of VWF depends on the multimer size: those freshly secreted from activated endothelial cells are ultra-large (UL) and overly adhesive, capable of forming spontaneous high strength bonds with the platelet receptor. In contrast, VWF multimers circulating in blood (pVWF) is required to be activated by high fluid shear stress or modulators in order to bind and aggregate platelets. Shear stress has previously been demonstrated to convert globular shape VWF multimers to elongated rope-like structures, but whether this structural change correlates with VWF adhesion activity remains largely unknown. We have showed that, upon secretion, ULVWF multimers form long string-like structures on activated endothelial cells that can be viewed under a regular light microscope, suggesting laterally association between ULVWF multimers. Furthermore, pVWF multimers can be induced to laterally associate with each other by shear stress, potentially resulting in the formation of VWF fibrils. We hypothesize that by forming laterally associated strings, ULVWF multimers require a greater physical force to unfold as compared to pVWF, which can be induced to form fibrillar structures that become more resistant to force-induced unfolding. Here, we present experimental data to support this hypothesis. First, we found that shear stress covalently aggregated pVWF multimers and the process was prevented rADAMTS-13 or disulfide reducing agents. VWF multimers were not cleaved by ADAMTS-13 under this experimental condition, suggesting a non-proteolytic activity. Second, when pVWF multimers are captured and subjected to physical pulling force on an atomic force microscope, the length of VWF multimers extended sequentially in response to increasing force. Plasma VWF multimers were unfolded at the peak forces of 115 pN, which increased to 153 pN after they were exposed to a pathological level of shear stress (100 dyn/cm2) for 3 min at 37°C. The peak force required to unfold pVWF after shear exposure (153 pN) was very similar to that of ULVWF multimers (152 pN). Third, recombinant (r) ADAMTS-13 did not reduce the peak force for unfolding pVWF multimers (115 pN) before but it lessened the increase in magnitude in the peak force required to unfold pVWF exposed to shear stress. This ADAMTS-13 activity was not inhibited by 5 mM of EDTA. Similarly, the peak force for unfolding ULVWF multimers was reduced from 152 pN to 127 pN when ULVWF was pulled in the presence of an equal molar concentration of rADAMTS-13. Taken together, these data demonstrate that shear stress significantly increases the peak force required to unfold pVWF multimers to the levels similar to ULVWF multimers, likely by promoting the formation of laterally associated VWF fibrils. ADAMTS-13 prevents the shear-induced increase in the peak force for unfolding sheared pVWF multimers, independent of the VWF proteolytic activity of the metalloprotease. The results reveal a mechanism for shear-induced VWF activation. They also suggest that ADAMTS-13 contains a non-proteolytic activity that plays a role in cleaving ULVWF strings and preventing pVWF multimers to be activated by lateral association induced by high fluid shear stresses.

Effects of Naturally Occurring Mutations in CUB-1 Domain on ADAMTS-13 Synthesis, Stability and Activity.

Upon stimulation, vascular endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and prothrombotic forms. The hyperactive ULVWF multimers are rapidly cleaved by the metalloprotease ADAMTS-13 to smaller forms that are hemostatically active, but no longer prothrombotic. Lack of this VWF- cleaving activity results in accumulation of ULVWF, which agglutinates platelets, leading to microvascular thrombosis as demonstrated in patients with thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 deficiency seen in TTP results from either mutations in the ADAMTS13 gene in familial cases or autoantibodies against the metalloprotease in acquired cases. ADAMTS-13 differs from other members of the family metalloproteases by containing two C-terminal CUB domains, which contain the binding site for VWF and whose mutations are associated with TTP. However, recombinant ADAMTS-13 without CUB domains remains active in vitro. To study the functional role of CUB domains, three naturally occurring mutations in the CUB-1 domain (C1213Y, W1245del and K1256frameshift, the latter two removed CUB-2 and the C-terminal part of CUB-1 domain) were generated by site-directed mutagenesis and expressed in the mammalian Hela cells. The release, stability, and activity of the mutants were then examined. We found that the mutations significantly reduced the release of recombinant ADMATS-13 into the culture medium, but not to the extracellular matrix. The intracellular pools of the recombinants detected in cell lysates were comparable to that of wild-type, indicating that the mutations affected the targeted secretion of the metalloprotease. When measured during a course of up to one month at −80°C, C1213Y and K1256framshift significantly accelerated, whereas W1245del delayed, degradation of the recombinants, which was inhibited by 5 mM of EDTA. The observation demonstrates that CUB domains are also critical for the structural stability of the metalloprotease. In comparison, the three mutants remained active in cleaving (UL)VWF under static and flow conditions. Consistent with the activity measurements, all three mutants bound immobilized VWF with similar kinetics in ELISA assay, indicating that the N-terminal sequence (C1192-D1212) of CUB-1, which is not affected by the mutations, contains the binding site for VWF. The conclusion is supported by our previous data that the CUB-1 synthetic peptides derived from the N-terminal region (H1196-P1220) blocked VWF-ADAMTS-13 interaction and cleavage of ULVWF under flow. Together, these results demonstrate that1.the CUB domains (primarily CUB-2 and the C-terminal part of CUB-1) are crucial for regulating the targeted ADAMTS-13 secretion and the stability of the metalloprotease;2.the N-terminal sequence of CUB-1 domain contains the VWF binding site that remains intact after deleting the rest of CUB domains; and3.the primary cause of ADAMTS-13 deficiency found in patients carrying these mutations is likely due to defective secretion of the metalloprotease.

Identification of ADAMTS13 Epitopes Required for Binding to von Willebrand Factor Using Lambda Phage Surface Display.

The metalloprotease ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. We planned to search core epitopes of ADAMTS13 that is required for its binding to VWF. We constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using immobilized VWF as a probe. After the first screening, the C-terminus of the spacer domain from Arg670 to Glu684 (termed as epitope-1) and the middle of the cysteine-rich domain from Arg484 to Arg507 (epitope-2) were determined as epitopes. When we added the synthetic epitope-1 peptide to the second screening, a new site, from Pro618 to Glu641 (epitope-3), was found in the middle of spacer domain. While the presence of synthetic epitope-2 peptide did not affect the subsequent screening, the presence of epitope-3 peptide enhanced the isolation of clones encoding epitope-1. These results suggest that ADAMTS13 epitopes-1, -2 and -3 may interact with each other for their binding to VWF. From screening in the presence of any combination or all of the three synthetic peptides, however, no new VWF binding site was uncovered. To examine the effect of divalent metal cations on the binding of ADAMTS13 epitopes to immobilized VWF, screening was carried out in the presence or absence of 5 mM of EDTA. No new epitope site was found. We next explored inhibitory effect of the synthetic epitope peptides on ADAMTS13 protease activity using recombinant ADAMTS13 and FRETS-VWF73 as a substrate. Synthetic epitopes-2 and -3 peptides markedly inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-1 peptide did not as efficiently as epitopes-2 and -3. The stronger inhibitory effect of epitope-3 peptide than that of epitope-1 peptide was confirmed by SDS-agarose gel electrophoresis analysis of cleavage products of denatured multimeric VWF molecules by recombinant ADAMTS13. This was consistent with the dissociation constants for the three synthetic peptides with immobilized VWF determined by surface plasmon resonance, in which epitopes-2 and -3 have higher affinities for VWF than that of epitope-1. The results described above suggest that ADAMTS13 may initially bind to immobilized VWF through the sites of epitope-1 and epitope-2 with relatively weak affinity. The binding of epitope-1 to VWF may subsequently induce the conformational change of VWF, thereby exposing a binding site for epitope-3 for the efficient catalytic cleavage of VWF by ADAMTS13.

Studies on the stability of protease from Bacillus sp. and its compatibility with commercial detergent

Enzymes, and particularly proteases, have become an important and indispensable part of industrial processes such as laundry detergents, pharmaceuticals and food products. Detergents such as Tide®, Ariel® and Biz® contain proteolytic enzymes, most of them produced by members of the genus Bacillus. This paper describes the compatibility of protease produced by the thermophilic Bacillus sp, with commercial laundry detergent. Stability studies indicated that this enzyme retained about 95% and 74% of its maximum activity after 1h at 60oC in the presence of glycine in combination with MnSO4 and CaCl2, respectively. No inhibitory effect was observed at 1.0-5.0 mM of EDTA. Triton X-100 inhibited the enzyme in all the concentrations tested. The enzyme was unstable in a 5% (v/v) concentration of peroxide solution. The protease retained more than 80% and 65% of its activity after 30 min incubation at 60oC in the presence of Tide® and Cheer® detergents, respectively. After supplementation of CaCl2 (10 mM) and glycine (1 mM), the enzyme in Tide® detergent retained more than 85% of its activity after 1h. Based on these findings, Bacillus sp. protease shows a good potential for application in laundry detergents.

Desenvolvimento de iniciadores para detecção e identificação de Diaporthe phaseolorum var. meridionalis em sementes de soja

Considerando a grande variabilidade apresentada pelo complexo Diaporthe/Phomopsis, os métodos rotineiros de sanidade de sementes, empregados na detecção do gênero Diaporthe, não são confiáveis visto que, para sua identificação são utilizadas as características morfológicas das colônias que se desenvolvem sobre as sementes. Diante disso, este trabalho teve como objetivo desenvolver iniciadores específicos, bem como um método confiável para detecção e identificação de Diaporthe phaseolorum var. meridionalis (Dphm), em sementes de soja. Amostra de sementes da cultivar IAS5, analisada previamente para sanidade, não portadoras de Diaporthe (SS) foram inoculadas com Dphm (SI), obtendo-se amostras com as seguintes proporções de SI:SS: 1:10, 1:50, 1:100, 1:200 e 1:400. Para a extração de DNA do micélio do patógeno das sementes foi necessário primeiro incubar as sementes por 7 dias, utilizando-se o método do papel de filtro modificado com 2,4 D e, em seguida submetê-las à lavagem em tampão de extração (1% de SDS, 10 mM de TRIS/HCL e 25 mM de EDTA) por 20 min, sob agitação a 100 rpm. Em seguida, alíquotas de 350 miL do sobrenadante foram transferidas para novos microtubos contendo 350 miL de resina de sílica gel da Wizard/Promega permanecendo em contacto com a mesma por 1 minuto. Os DNAs extraídos foram utilizados como molde na reação de PCR com os iniciadores: DphLe (TCG GCC TTG GAA GTA GAA AG) e DphRi (ACT GAA TGC GTT GCG ATT CT) Utilizando-se estes iniciadores, foi possível detectar a presença de D. phaseolorum var meridionalis em sementes de soja, na proporção de uma semente infectada com o patógeno em amostras de 400 sementes (0,25% de incidência), utilizando-se o método do papel de filtro modificado com 2,4 D associado à técnica de PCR.

Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616

A maltogenic amylase-producing thermophilic strain WPD616, assigned to Bacillus sp. WPD616 based on 16S rRNA sequence, was isolated from a deep-sea hydrothermal field in west Pacific. Subsequently, a maltogenic amylase gene encoding 588 amino acids from this isolate was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The results showed that the recombinant maltogenic amylase had an activity optimum at 50 °C and pH at 6.0. It was active up to 70 °C at pH 6.0 and stable at pHs ranging from 6.0 to 8.0. The recombinant enzyme was active when Chaps, DTT and Tween 20 (0.1% or 1%) were used. However, it can be partially inhibited by 1 mM of EDTA, PMSF or SDS, as well as 0.1% of Triton X-100 or 2-ME, and completely inhibited by 10 mM of PMSF and SDS (10 mM). Its catalytic function was stable in the presence of Li + and K + (1 mM or 10 mM), but its activity decreased when Ba 2+, Ca 2+, Mg 2+, Mn 2+ (1 mM or 10 mM) and Fe 2+ (1 mM) were used. In the presence of Zn 2+, Cu 2, Fe 3+ (1 mM and 10 mM) and Fe 2+ (10 mM), no activity was detected.