Inhibitor Of DNase Research Articles

Study of Plasmid Profiling of Clostridium perfringens Isolated from Small Ruminants of Odisha

In the present study 209 intestinal and faecal samples of small ruminants were collected from different farms and localities in and around Bhubaneswar, Odisha. Out of which 159 samples were of goats and 50 samples of sheep, it includes both intestinal and faecal samples. The collected samples were processed for plasmid profiling of C. perfringens. A total of 19 samples were found positive for C. perfringens indicating the prevalence of 9.09% in small ruminant population of Odisha. In order to study the epidemiological strain variation, all the present isolates were subjected to plasmid profiling. Plasmid profiling of the local isolates is an important tool for strain related identification of disease in sheep and goat. In this study plasmid DNA yield was low, as for high yield large amount of fresh C. perfringens culture was needed, but an attempt has been carried out for plasmid curing. Effect of various parameters like different percentage of gel, running buffer and temperature were done and it was found that 0.7% agarose gel, TBE buffer at 30oC of temperature were optimum condition for plasmid profiling. The present study showed that DNase activity remains a block in the plasmid isolation and profiling of Clostridial species. Therefore need for a DNase inhibitor that may increase the efficacy of plasmid curing.

Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury

BackgroundCirculating cell‐free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury.MethodsPatients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months.ResultsAnalysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1–28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D‐binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels.ConclusionThe results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA‐induced host tissue damage and thrombosis.

Characteristics and influencing factors of amyloid fibers in S. mutans biofilm

There are signs that amyloid fibers exist in Streptococcus mutans biofilm recently. However, the characteristics of amyloid fibers and fibrillation influencing factors are unknown. In this study, we firstly used transmission electron microscopy (TEM) and atomic force microscopy (AFM) to observe the morphology of amyloid fibers in S. mutans. Then the extracted amyloid fibers from biofilm were studied for their characteristics. Further, the influencing factors, PH, temperature and eDNA, were investigated. Results showed there were mainly two morphologies of amyloid fibers in S. mutans, different in width. Amyloid fibers inhibitor-EGCG obviously destroyed biofilm at different stages, which is dose-dependent. The amount of amyloid fibers positively correlated with biofilm biomass in clinical isolates. Acidic pH and high temperature obviously accelerated amyloid fibrillation. During amyloid fibrillation, amyloid growth morphologies were observed by TEM and results showed two growth morphologies. Amyloid fibers formed complex with eDNA, which we call (a)eDNA. The molecular weight of (a)eDNA was similar to genomic DNA, greatly larger than that of eDNA in matrix. Combined use of DNase I and EGCG was more efficiently in inhibiting amyloid fibers and biofilm biomass. In conclusion, amyloid fibers are the crucial structures for S. mutans biofilm formation, showing two types of morphology. Acidic pH and temperature can obviously accelerate amyloid fibrillation. Amyloid fibers form complex with (a)eDNA and combined use of DNase and amyloid fiber inhibitor is more efficiently in inhibiting S. mutans biofilm formation.

Induction of Melanoma Cell-Selective Apoptosis Using Anti-HER2 Antibody-Conjugated Gold Nanoparticles.

PurposeThis study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2).Materials and MethodsFollowing GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting.ResultsG361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation.ConclusionGNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.

Inhibition of DNase I Enzyme with Nickel(II) Triphenylphosphine Complexes Incorporating Tridentate Schiff Base Ligands in Vitro

The nickel(II) complexes containing 3-methoxy-salicylaldehyde-N4-R thiosemicarbazones (R:-H2,-propyl)with triphenylphosphine coligands have been synthesized. The structure of Ni(II)-centered metal complex was approved by elemental analysis and melting point. The solid-state structure of complex 2 bearing PPh3 as co-ligand was clarified by single crystal X-ray crystallography, which revealed square planar geometry around Ni(II) ion. Thiosemicarbazone ligands are coordinated by ONS mode to nickel(II). The potential of these complexes to inhibit the DNase I enzyme, which uses DNA as a substrate, was investigated in vitro. The results revealed that the compounds inhibited the DNase enzyme in directly and/or indirectly (by masking of DNA molecules) at ≥0.1 µg/ml concentrations in vitro.

In silico prediction of active site and in vitro DNase and RNase activities of Helicoverpa-inducible pathogenesis related-4 protein from Cicer arietinum

Plants are endowed with an innate immune system, which enables them to protect themselves from pest and pathogen. The participation of pathogenesis-related (PR) proteins is one of the most crucial events of inducible plant defense response. Herein, we report the characterization of CaHaPR-4, a Helicoverpa-inducible class II PR-4 protein from chickpea. Bioinformatic analysis of CaHaPR-4 protein indicated the presence of a signal peptide, barwin domain but it lacks the chitin-binding site/hevein domain. The recombinant CaHaPR-4 is bestowed with RNase and bivalent ion-dependent DNase activity. Further, the RNA and DNA binding sites were identified and confirmed by analyzing interactions between mutated CaHaPR-4 with the altered active site and ribonuclease inhibitor, 5′ADP and DNase inhibitor, 2‑nitro‑5‑thiocyanobenzoic acid (NTCB) using 3D modeling and docking studies. Moreover, CaHaPR-4 shows antifungal activity as well as growth inhibiting properties against neonatal podborer larvae. To the best of our knowledge, this is the first report of a PR-4 showing RNase, DNase, antifungal and most importantly insect growth inhibiting properties against Helicoverpa armigera simultaneously.

Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells.

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 μM TS primer (5'-AATCCGTCGAGCAGAGTT-3') and 0.04 μM ACX primer (5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3'). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration.

Improved genetic transformation of Synechococcus elongatus PCC 7942 using linear DNA fragments in association with a DNase inhibitor

The genetic manipulation in many cyanobacterial strains is challenging yet. Thus, the development of new transformation protocols is desirable to facilitate the genetic engineering in cyanobacteria. Transformations using linear fragments yielded by PCR have advantages such as: less laborious methodology, faster procedure, low cost and unnecessary cloning steps. However, some strains presence extracellular nucleases, which reduce the efficiency in obtaining transformants. In this study, we demonstrate an improved protocol for genetic transformation in Synechococcus elongatus PCC 7942 using linear fragments employing EDTA-mediated inhibition of DNases. To conduct the transformation, linear PCR products containing the spectinomycin antibiotic resistance gene were employed. As result, 40mM EDTA treatment increased the number of transformants obtained by eightfold in comparison to the conventional protocol using plasmid DNA. Thus, the application of exonuclease inhibitors can be considered a relevant improvement to manipulate cyanobacteria in a more efficient, faster way and as a low-cost alternative. This protocol must be helpful for other strains of cyanobacteria.

Abstract 12983: mtDNA Damage in Concert With PCSK9 Expression Induces Apoptosis in Vascular Smooth Muscle Cells

Introduction: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in inducing hypercholesterolemia and associated cardiovascular diseases. Autophagy is considered a protective and adaptive mechanism to promote cell survival, while apoptosis represents cell death program designed to remove injured cells. Exposure of vascular smooth muscle cells (SMCs) to noxious stimuli such as LPS or ox-LDL induces PCSK9 expression and apoptosis. Methods and Results: Treatment of human primary vascular SMCs to 20 ng/ml LPS significantly induced both mtDNA damage and PCSK9 expression. mtDNA damage induction with the autophagy inhibitor 3-methyladenine enhanced PCSK9 expression, whereas mtDNA damage inhibition with the autophagy inducer rapamycin reduced PCSK9 expression, indicating an interplay between autophagy, mtDNA damage and PCSK9. Inhibition of DNase II (siRNA) for reduced the extent of mtDNA damage and PCSK9 expression confirmed the role of mtDNA damage in PCSK9 expression. Next, we investigated the role of PCSK9 in the development of apoptosis. We observed that PCSK9 upregulation by hPCSK9 treatment induced and PCSK9 inhibition by siRNA transfection reduced apoptosis of SMCs (measured as TUNEL staining and expression of Bax, Bcl2 and cleaved caspase-3). Conclusions: These observations provide a compelling evidence for the role of mtDNA damage in PCSK9 release. Both collectively induce SMC apoptosis during inflammatory states characteristic of cardiovascular diseases, such as atherosclerosis.

Facile mechanical shaking method is an improved isolation approach for islet preparation and transplantation.

Successful islet isolation is crucial for islet transplantation and cell treatment for type 1 diabetes. Current isolation methods are able to obtain 500–1,000 islets per rat, which results in a waste of ≥50% of total islets. In the present study, a facile mechanical shaking method for improving islet yield (up to 1,500 per rat) was developed and summarized, which was demonstrated to be more effective than the existing well-established stationary method. The present results showed that isolated islets have a maximum yield of 1,326±152 when shaking for 15 min for the fully-cannulated pancreas. For both fully-cannulated and half-cannulated pancreas in the presence of rat DNAse inhibitor, the optimal shaking time was amended to 20 min with a further increased yield of 1,344±134 and 1,286±124 islets, respectively. Furthermore, the majority of the isolated islets were morphologically intact with a well-defined surface and almost no central necrotic zone, which suggested that the condition of islets obtained via the mechanical shaking method was consistent with the stationary method. Islet size distribution was also calculated and it was demonstrated that islets from the stationary method exhibited the same size distribution as the non-cannulated group, which had more larger islets than the fully-cannulated and half-cannulated groups isolated via the shaking method. In addition, the results of glucose challenge showed that the refraction index of each group was >2.5, which indicated the well-preserved function of isolated islets. Furthermore, the transplanted islets exhibited a therapeutic effect after 1 day of transplantation; however, they failed to control blood glucose levels after ~7 days of transplantation. In conclusion, these results demonstrated that the facile mechanical shaking method may markedly improve the yield of rat islet isolation, and in vitro and in vivo investigation demonstrated the well-preserved function of isolated islets in the control of blood glucose. Therefore, the facile mechanical shaking method may be an alternative improved procedure to obtain higher islet yield for islet preparation and transplantation in the treatment of type 1 diabetes.

"The NET Outcome": Are Neutrophil Extracellular Traps of Any Relevance to the Pathophysiology of Autoimmune Disorders in Childhood?

Neutrophil extracellular trap (NET) formation represents a form of cell death distinct from apoptosis or necrosis, by which invading pathogens are simultaneously entangled and potentially eliminated. Increased NET formation is observed in systemic lupus erythematosus (SLE), rheumatoid arthritis, antineutrophil cytoplasmic antibody-associated small vessel vasculitis, antiphospholipid antibody syndrome (APS), and psoriasis. NETs contribute to the pathogenesis of autoimmunity by exposing cryptic autoepitopes, which may facilitate the generation of autoantibodies, induce the production of interferons, and activate the complement cascade. In SLE, augmented disease activity and renal disease are associated with increased NET formation, so that NETs could serve as a marker for the monitoring of disease activity. NETs can additionally cause endothelial cell damage and death and stimulate inflammation in atheromatous plaques, adding to the accelerated atherosclerosis witnessed in autoimmune disease. Since NETs induce production of interferons, assessing the extent of NET formation might facilitate the prediction of IFN-alpha levels and identification of SLE patients with presumably better responses to anti-IFN-alpha therapies or other novel therapeutic concepts, such as N-acetyl-cysteine and inhibitors of DNase 1 and peptidylarginine deiminase 4 (PAD4), which also target NETs. In summary, the study of NETs provides a novel approach to the understanding of autoimmune disease pathogenesis in childhood and opens new vistas in the development of sensitive disease markers and targeted therapies.

Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma - Tissue Microarray of Human Samples.

Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples

ObjetivesThe extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool. Design and methodsEDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase. ResultsThe endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma. ConclusionThe exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma.

Novel High-Throughput Deoxyribonuclease 1 Assay

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

Deoxyribonuclease inhibitors.

Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or modify their activities. This review focuses on DNase inhibitors. Some DNase inhibitors have been isolated from various natural sources, such as humans, animals (beef, calf, rabbit and rat), plants (Nicotiana tabacum), and microorganisms (some Streptomyces and Adenovirus species, Micromonospora echinospora and Escherichia coli), while others have been obtained by chemical synthesis. They differ in chemical structure (various proteins, nucleotides, anthracycline and aminoglycoside antibiotics, synthetic organic and inorganic compounds) and mechanism of action (forming complexes with DNases or DNA). Some of the inhibitors are specific toward only one type of DNase, while others are active towards two or more. Physico-chemical properties of DNase inhibitors are calculated using the Molinspiration tool and most of them meetall criteria for good solubility and permeability. DNase inhibitors may be used as pharmaceuticals for preventing, monitoring and treating various diseases.

Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation.

BackgroundBiofilm formation by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity.ResultsInitially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24 h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (∆cht2, ∆cht3) and double knockout (∆cht2/∆cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold.ConclusionsCollectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0303-6) contains supplementary material, which is available to authorized users.

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis.

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.

Labware, Lab Supplies, and Microplates

Labware, Lab Supplies, and Microplates

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.

Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA-membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored. To visualize the DNA-membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA-membrane interaction was observed with HV + LV pulses. The formation of the DNA-membrane complex depended on the Mg(2+) concentration, whereas DNase inhibitor did not affect gene expression. Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.