Oligomer based real-time detection of microorganisms producing nuclease enzymes.

In this study, we provide a method using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases in real time, while growing them in culture media. The detection of such microorganisms was possible in a short period of time, as short as 10 minutes up to a maximum of 8 hours, depending on the bacterial density. We also showed the suitability of this new method for determination of minimum inhibitory concentration (MIC) in culture media in a very short period of time, compared to conventional methods. We believe that it can make a significant contribution to gain new insights for analysis of complex materials such as clinical samples, food samples and environmental samples.

Hairpin loop-enhanced fluorescent copper nanoclusters and application in S1 nuclease detection.

Novel highly fluorescent copper nanoclusters (CuNCs) were prepared by using 24 adenine-thymine pair dsDNA (AT24) with six-base (X6) loops (AT24-X6-hairpin DNA) as an effective template. The AT24 double strand stem serves as a template for CuNC formation, and the six-base sequence loop acts as specific regions to enhance the fluorescence intensity of CuNCs. Relative to the AT24-CuNCs, AT24-X6-hairpin CuNCs have greater fluorescence (5 times enhancement). What's more, the influence of the hairpin loop with different base types and base numbers on the fluorescence of CuNCs was first proposed and investigated. By choosing an AT24 double strand stem, any types of base loops can enhance the fluorescence of CuNCs. However, the fluorescence enhancement would be reduced with an increasing number of hairpin loop sequences. Besides this, the successful detection of S1 nuclease demonstrates its potential to be a new and robust fluorescent probe for sensing applications.

Single-Stranded DNA Assisted Cell Penetrating Peptide-DNA Conjugation Strategy for Intracellular Imaging of Nucleases.

Cell penetrating peptides (CPPs) are very useful tools for delivery of DNA molecules into living cells without damaging the cell membranes. However, covalent conjugation of DNAs to CPPs is technically difficult, and the reactions between DNA and target nucleases are also liable to be affected by the cationic CPP molecules. In this work, we demonstrate that the electrostatic interactions between CPPs and single-stranded DNA (ssDNA) were stronger than those between CPP and double-stranded DNA (dsDNA). Taking advantage of this property, we developed an ssDNA protected CPP-DNA fluorescent probe which allowed for noninvasive and efficient cellular uptake and rapid imaging of target nucleases in living cells. The probe is highly sensitive and selective. This work represents the first example of using CPP-DNA conjugate to deliver DNA fluorescent probes for in situ imaging of nucleases within cells. The developed approach also holds great potential for the cellular delivery of other nucleic acid molecules for diagnosis or therapeutics purposes.

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

DNA sensor's selectivity enhancement and protection from contaminating nucleases due to a hydrated ionic liquid.

The thermodynamic stability of certain mismatched base pairs has made the development of DNA sequence sensing systems challenging. Thus, the stability of fully matched and mismatched DNA oligonucleotides in the hydrated ionic liquid choline dihydrogen phosphate (choline dhp) was investigated. Mismatched base pairs were significantly destabilized in choline dhp relative to those in aqueous buffer. A molecular beacon that forms a triplex with a conserved HIV-1 sequence was then designed and tested in choline dhp. The molecular beacon specifically detected the target duplex via triplex formation at concentrations as low as 1 pmol per 10 µL with 10,000-fold sequence selectivity. Moreover, the molecular beacon was protected from a contaminating nuclease in choline dhp, and DNAs in aqueous solutions were not sufficiently stable for practical use.

[Cytochemical localization and properties of selected nucleolytic enzymes]. / Cytochemiczna lokalizacja i wlasciwosci wybranych enzymów nukleolitycznych.

In the article there are shortly outlined studies on cytochemical localization of selected nucleolytic enzymes carried out between 1957-1986 by David Shugar and his coworkers. The histochemical localization of several nucleolytic enzymes in animal and plant tissues was determined by synthesis of specific substrates, alpha-naphthyl esters of 5'- and 3'-nucleotides and their derivatives. In rat tissues phosphodiesterase I was localized in the plasma membrane whereas phosphodiesterase II in the lizosomes, reflecting their physiological roles. The localization of pancreatic type ribonuclease in animal tissues was determined, indicating its role in extracellular digestion. Plant nucleotide pyrophosphatase was localized in several tissues, purified to near homogeneity from potato tubers and its properties and substrate specificity were determined. Application of this enzyme for removal of m7GMP from the "cap" of eukaryotic mRNA allowed to elucidate the role of "cap" in mRNA binding to ribosomes in the process of translation. Furthermore, cyclic nucleotide phosphodiesterase was isolated from potato tubers and its physicochemical properties, oligomeric structure and substrate specificity were elucidated.

Highly sensitive determination of ssDNA and real-time sensing of nuclease activity and inhibition based on the controlled self-assembly of a 9,10-distyrylanthracene probe.

We report here a fluorescent biosensor for highly sensitive determination of single-stranded DNA (ssDNA) with remarkable fluorescence enhancement and label-free sensing of S1 nuclease activity and inhibition in real time based on ssDNA-controlled self-assembly of a 9,10-distyrylanthracene (DSA) probe with the aggregation-induced emission (AIE) property, thereby avoiding a sophisticated fabrication process and aggregation-caused quenching (ACQ) effect. Compared with previous technologies, this assay has some advantages. First, since the DSA probe can be synthesized through a simple and effective synthetic route and the sensing technology adopts the unlabelled ssDNA, this biosensor shows advantages of simplicity and cost efficiency. Besides, for the determination of ssDNA, S1 nuclease, and inhibitor, the DSA-based probe provides high sensitivity and a good linear relationship due to the AIE property. As a result, we determined the DNA 24-mer concentration as low as 150 pM, and we are able to detect ssDNA lengths with a linear range from 6mer to 24mer (R = 0.998) as well as DNA 24-mer concentrations with a linear range from 0 to 200 nM (R = 0.998) and S1 nuclease concentrations with a linear range from 6 to 32 U ml(-1) (R = 0.995), respectively. Moreover, the fluorescent intensity with various concentrations of S1 nuclease becomes highly discriminating after 3-16 min. Thus, it is possible to detect nuclease activity within 3-16 min, which demonstrates another advantage of a quick response of the present biosensor system.

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application.

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 µg ml(-1)), amoxicillin/clavulanic acid (30 µg ml(-1)) and cephalothin (30 µg ml(-1)) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

[Pathogenicity factors of enterococci of human intestinal microflora].

AIM: Characteristic of pathogenicity factors of enterococci isolated from human feces. MATERIALS AND METHODS: Production of hemolysin, gelatinase and DNase was determined in 161 enterococci cultures. RESULTS: Hemolytic activity detected in 14.9 +/- 2.8% of the studied cultures was the most prevalent characteristic; 22 of 24 hemolytic strains belonged to E. faecalis species. Human erythrocyte lysis was also caused by E. faecium and E. durans cultures (1 strain each). Other pathogenicity factors were detected solely in E. faecalis species members. Enterococci proteolytic activity associated with gelatinase enzyme production manifested on various substrates--both gelatin and milk. This property was detected in 7.5 +/- 2.1% cultures. Deoxyribonuclease was detected in 1 (1.2 +/- 0.9%) E. faecalis strain. A number of E. faecalis cultures possessing hemolytic activity additionally hydrolyzed gelatin (22.7 +/- 8.9% strains) and DNA (4.5 +/- 4.4% isolates). CONCLUSION: Though pathogenicity factors occur in enterococci of intestine microflora relatively rarely, separate cultures with expression of 2 or more pathogenicity factors may be essential in the development of endogenous infections especially in immune compromised patients.

[Persistence properties of Corynebacterium non diphtheriae circulating in Rostov-on-Don and Rostov region].

AIM: Characterize persistence properties and antibiotic sensitivity of Corynebacterium non diphtheriae circulating in Rostov-on-Don and Rostov Region. MATERIALS AND METHODS: DNase, anti-immunoglobulin activity, hemagglutinating activity, antagonistic properties and antibiotic sensitivity of Corynebacterium non diphtheriae strains isolated from patients with inflammatory diseases of urogenital tract, pregnant women and individuals undertaking prophylactic examination were studied. RESULTS: Lack of antagonistic interactions of C. non diphtheriae with members of opportunistic microorganisms, high anti-immunoglobulin activity against IgM and IgA, lack of hemagglutinating activity were established. Cefazolin and benzylpenicillin had the highest antibacterial activity against C. non diphtheriae, azithromycin and lincomycin--the lowest. CONCLUSION: Population level of C. non diphtheriae during urogenital tract disease diagnostics is necessary. Determination of their sensitivity to a wide specter of antibacterial preparations is reasonable for optimal selection of therapeutic agents.

[The significance of some potentially pathogenic microorganisms in occurrence of food toxicosis. Part 1. S. aureus and staphylococcal enterotoxins].

The data on the nomenclature, classification and properties of staphylococci and staphylococcal enterotoxins produced by them are presented. The analysis of cultural and biochemical properties of 137 strains of staphylococci isolated from raw milk and "Russian" cheese was performed. The high degree of correlation between the ability of S. aureus produce enterotoxins and the presence of enzymes coagulase, thermostable DNase, and other factors of pathogenicity is established.

Mass isolation and culture of rat kupffer cells.

Collagenase perfusion of the liver followed by pronase treatment of the cell suspension thus obtained gave a quantitative recovery of viable nonparenchymal liver cells (NPC). From these NPC, Kupffer (K) cells can be purified by attachment to tissue culture dishes. Tail vein injection of carbon 1-2 h before liver perfusion permitted stepwise calculation as well as visualization of carbon-containing K cells. When these K cells have been put into tissue culture medium with serum and incubated overnight, they exhibit typical macrophage characteristics. Phase-contrast and transmission electron microscopy showed typical macrophage morphology and scanning electron microscopy revealed well-spread cells with cytoplasmic projections and ruffled membranes. Endocytosis studies using radioactive colloidal gold and inert latex particles also indicated that these cells are highly active in pinocytosis and phagocytosis. Further characterization of K cells is the identification of Fc receptor on their membranes. Studies on lysosomal enzymes showed that purified K cells possess higher specific activities in beta-glucuronidase, acid DNase, and cathepsin D than in purified parenchymal cells.

Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii.

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2 mm) than among environmental strains (average radius of 2.9 mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31 kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans-C. gattii species complex pathogenicity.

Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells.

The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.

Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes.

The response of rat liver lysosomes to an intraperitoneal injection of glucagon has been evaluated from studies on the mechanical fragility, osmotic sensitivity, and sedimentation properties of these subcellular particles. It has been found that about (1/2) hr after the injection of glucagon the hepatic lysosomes exhibit a fairly sudden increase in their sensitivity to mechanical stresses and to exposure to a decreased osmotic pressure. At the same time, their sedimentation properties undergo complex changes characterized mainly by a significant increase in the sedimentation coefficient of a considerable proportion of the total particles. In addition, glucagon causes an increase in the proportion of slowly sedimenting particles, with the result that the distribution of sedimentation coefficients within the total population tends to become bimodal. The latter change is more pronounced for acid phosphatase, less so for cathepsin D, and barely detectable for acid deoxyribonuclease. All these modifications are maximal between 45 and 90 min after injection and regress to normal within approximately 4 hr. With the exception of the increase in the slow component, for which no explanation can be advanced at the present time, they are consistent with the hypothesis that glucagon causes an increase in lysosomal size, and may be related to the autophagic-vacuole formation known to occur after glucagon administration.

Analytical fractionation of homogenates from cultured rat embryo fibroblasts.

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-beta-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.

Concomitant synthesis of membrane protein and exportable protein of the secretory granule in rat parotid gland.

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-(14)C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears.

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, beta-galactosidase, beta-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.

Neuraminidase activity in hela cells: effect of hydrocortisone.

Homogenates of HeLa cells contain neuraminidase activity. This enzyme is particle-bound, and it has a pH optimum of 4.2. Hydrocortisone-regulated cells contain two to three times as much neuraminidase as the corresponding controls. The hydrocortisone treatment also causes an increase in the cell content of beta-glucuronidase and acid deoxyribonuclease.