Administración intrapleural secuencial de agentes fibrinolíticos y dornasa alfa en el empiema. Protocolo de uso clínico en base a su estabilidad fisicoquímica / Sequential intrapleural administration of fibrinolytic drugs and dornase alfa for empyema management. Treatment protocol based on its physicochemical stability

Objetivo: La administración intrapleural de fibrinolíticos y dornasa alfa ha demostrado en estudios aleatorizados ser capaz de disminuir la necesidad de desbridamiento quirúrgico del empiema y los días de estancia media hospitalaria. Sin embargo, su aplicación en práctica clínica es limitada, probablemente debido a la falta de protocolos que simplifiquen su administración. El presente estudio tiene como objetivo analizar la estabilidad fisicoquímica de la administración simultánea de uroquinasa y dornasa alfa para el posterior desarrollo de un protocolo de uso en práctica clínica. Método: Ensayo de estabilidad in vitro de uroquinasa, dornasa alfa y la mezcla de ambos. Se evaluó su estabilidad como (i) ausencia de partículas, (ii) variación de color y (iii) cambios de pH a tiempos 0,30 minutos, 1, 2 y 4 horas a 37°C. Cada muestra se preparó y analizó por triplicado. Resultados: Las soluciones individuales de uroquinasa y dornasa alfa mostraron cambios ligeros del pH, sin cambios en su color ni presencia de partículas en suspensión. La mezcla de uroquinasa y dornasa alfa no fue estable transcurridas 2 horas, mostrando turbidez por la floculación y separación de fases con formación de precipitado a las 4 horas. Se desarrolló un protocolo de uso clínico basado en la administración secuencial de uroquinasa y dornasa alfa, ya que no fue posible garantizar la estabilidad fisicoquímica de la administración simultánea de ambos fármacos. Conclusiones: Los datos de estabilidad fisicoquímica no permiten asegurar la administración simultánea de ambos fármacos de manera segura y eficaz, por lo que se propone un protocolo de administración secuencial

Objective: Intrapleural administration of fibrinolytics and dornase alfa has been shown in randomized studies to be able to reduce both the need for surgical debridement of empyema and the average hospital stay. However, its application in clinical practice is limited, probably due to the lack of protocols that simplify its administration. The present study aims to analyze the physicochemical stability of the simultaneous urokinase and dornase alfa administration for the subsequent development of a clinical practice use protocol. Method: In vitro stability test of urokinase, dornase alfa and a combination of both. Its stability was evaluated as (i) absence of particles, (ii) color variation and (iii) pH changes at times 0,30 minutes, 1,2 and 4 hours at 37°C. Each sample was prepared and analyzed in triplicate. Results: Individual solutions of urokinase and dornase alfa showed slight changes in pH, finding no changes in either color or presence of suspended particles. The urokinase and dornase alfa combination was not stable after 2 hours, when turbidity emerged due to flocculation and phase separation. After 4 hours, precipitate formation was found. A protocol for clinical use was developed based on urokinase and dornase alfa sequential administration, since it was not possible to guarantee the physicochemical stability of the simultaneous administration of both drugs. Conclusions: The physicochemical stability data obtained does not allow to ensure a simultaneous administration of both drugs in a safe and effective way, thus a sequential administration protocol is proposed

Effects of Lyse-It on endonuclease fragmentation, function and activity.

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.

Preparation and gene delivery of alkaline amino acids-based cationic liposomes.

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or L-arginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR.

BACKGROUND: Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. RESULTS: We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. CONCLUSION: Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes.

The protein transduction domain (PTD) of the HIV-1 Tat protein can facilitate the cellular and nuclear uptake of macromolecular particles. Here, we demonstrate that incorporation without covalent linkage of a 17-amino acid PTD peptide into gene delivery lipoplexes improves gene transfer. Tat/Liposome/DNA (TLD) transfection, as evaluated by Fluorescence Activated Cell Scan analysis of a Green Fluorescence Protein expression plasmid, enabled transfection of highly recalcitrant primary cells in the form of air/liquid interface cultures of sheep tracheal epithelium. Treatment with chloroquine increased, and incubation at low temperature decreased, TLD transfection, suggesting that the endocytosis uptake pathway is involved.

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA.

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.

Modified linear polyethylenimine-cholesterol conjugates for DNA complexation.

Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M(w) 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of approximately 250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.

Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.

Gelsolin activates DNase I in vitro and cystic fibrosis sputum.

Because actin can form a complex in vitro containing both gelsolin and DNase I, gelsolin and DNase I have been assumed to bind independently to actin. Although this assumption is consistent with the known crystalline structures of gelsolin with one actin and of actin with DNase I, which suggest that the binding sites on actin for both gelsolin and DNase I are distinct and separate, we propose that a second actin binding site on gelsolin competes with DNase I for actin. Since actin is an inhibitor of DNase I, competition at the second binding site results in activation of DNase I by gelsolin. Covalent cross-linking experiments confirm that DNase I prevents dimerization of actin by gelsolin, consistent with displacement of one actin from gelsolin by DNase I. Activation of DNase I by gelsolin is a novel function for a cytoskeletal protein and could have broad implications for biology, such as a role in initiating apoptosis. These results also may explain why both gelsolin and DNase I decrease sputum viscosity in cystic fibrosis (CF). While the activity of DNase I had originally been attributed to fragmentation of DNA, subsequent data suggested that both gelsolin and DNase I may affect viscosity by depolymerizing filamentous actin. The current results alternatively suggest aht dissociation of the actin-DNase I complex by gelsolin in CF sputum results in activation of the nuclease activity of constitutive DNase I. The nuclease activity of DNase I alone is therefore sufficient to explain the effects of both gelsolin and DNase I on CF sputum.

A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli.

The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli. Leucine modulates Lrp regulation of leucine-responsive operons. The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region. To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed. The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription. Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA. To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated. The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions. Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription. Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex.

Rapid induction of Fas antigen mRNA expression in vivo by cycloheximide.

The effect of administration into rats of cycloheximide on the expression of genes, such as tissue transglutaminase, testosterone-repressed prostate message-2, Fas antigen, bcl-2, DNase I, and poly(ADP-ribose) polymerase, which were believed to be involved in the mechanism of apoptosis, was studied. While the effect of cycloheximide on the expression of genes other than Fas antigen was modest, only the expression of Fas antigen was elevated rapidly in most of the organs examined. A possible direct effect of cycloheximide on cells per se to induce Fas antigen mRNA expression was demonstrated by the tissue culture study using L929 fibroblast cells, although the magnitude of the induction detected in vitro was small compared with that in vivo. This induction of Fas antigen mRNA by cycloheximide is a first report on the modulation of Fas antigen mRNA expression in vivo.

Genetic control of urinary deoxyribonuclease I (DNase I) activity levels in mice.

Genetic factors controlling the deoxyribonuclease I (DNase I) activity level were examined in mice. A survey of inbred strains of mice revealed genetic variation in urinary and kidney DNase I activity levels, though a sex difference, males having significantly higher DNase I activity levels than females, was observed in all mouse strains tested. The sex difference in the urinary DNase I activity level was eliminated by testosterone administration to females or gonadectomy to males. The urinary DNase I level was closely correlated to that of the kidneys but no relationship between serum and urinary DNase I activity suggests that the production ratio of DNase I in the kidneys is responsible for strain variation in urinary DNase I levels. Inheritance of quantitative variation of urinary DNase I activity levels was studied by a test cross. The segregation ratio of backcross progenies fitted the model showing that urinary DNase I activity level was controlled by an autosomal single locus, Dna 1 (chi 2 = 0.1053, P > 0.90). The allele Dna 1 ra determines high DNase I inducibility in the kidneys and occurs in BALB/c, C3H/He and A/J strains. The allele Dna 1 rb determines low DNase I inducibility in the kidneys and occurs in DBA/2 and C57BL/6 strains.

Chemoreceptor-mediated polymerization and depolymerization of actin in hair bundles of sea anemones.

Hair bundles located on tentacles of sea anemones are morphodynamic mechanoreceptors employed to regulate discharge of nematocysts into swimming prey. Activation of chemoreceptors for N-acetylated sugars is known to induce anemone hair bundles to elongate while shifting discharge to lower frequencies matching those produced by calmly swimming prey. In the continued presence of N-acetylated sugars, activation of proline receptors is known to induce hair bundles to shorten while shifting nematocyst discharge to higher frequencies presumed to correspond to movements produced by wounded, struggling prey. In the present study, N-acetylneuraminic acid (NANA) causes stereocilia to become more intensely fluorescent in confocal optical sections of phalloidin-stained specimens, suggesting that receptors for N-acetylated sugars initiate processes to increase the density of F-actin within stereocilia. Computer analysis of electron micrographs is consistent with this interpretation for large diameter stereocilia but not for small diameter stereocilia. In the continued presence of NANA, proline causes fluorescence intensity of phalloidin to decrease to or below control levels. DNaseI uniformly stains large diameter stereocilia, suggesting that these stereocilia contain a pool of G-actin. Fluorescence intensity of DNaseI in stereocilia is significantly less bright in specimens exposed to NANA alone than in specimens exposed to proline in the continued presence of NANA. It appears that whereas activated receptors for NANA induce G-actin to polymerize in large diameter stereocilia, activated receptors for proline induce F-actin to depolymerize, restoring G-actin pools.

[DNAse-I inhibition by the chromatin proteins of the brain cells in rats]. / Ingibirovanie DNKazy-I belkami khromatina kletok golovnogo mozga krys.

The effect of protein extracted from nuclei of brain cells on the reaction of DNA-ethidium bromide complex interaction with DNAase I and Bac. subtilis nuclease was studied. The nuclei were isolated from the brain of Wistar male rats weighting 180-200 g. The fluorescence was recorded on a Hitachi MPF-44B spectrofluorimeter. Proteins extracted from the nuclei with 0.6 NaCl substantially inhibited the DNAase I but not Bac. subtilis nuclease activity. Other studied fractions of nuclei lacked the capacity for inhibiting DNAase I.

Influence of suramin on some DNA-directed enzymes and primary cultures of chicken embryo and rat cells.

The influence of suramin on O6-alkylguanine-DNA alkyltransferase (AT), DNase I and poly(adenosine diphosphate ribose)polymerase (PADPR) as well as on primary cultures of rat and chick embryo cells was examined by using some short-term tests. AT and DNase I were inhibited by suramin in a dose-dependent manner (DE50 = 65 and 100 micrograms/ml, respectively). PADPR activity was increased over a concentration range of 40-320 micrograms/ml. At concentrations above 40 micrograms/ml suramin decreased scheduled and unscheduled DNA synthesis. At doses of below 20 micrograms/ml the substance slightly stimulated unscheduled DNA synthesis in embryonic cells. Suramin enhanced nucleoid sedimentation and diminished the viscosity of alkaline cell lysates. From the present results it is concluded that suramin, at clinically relevant concentrations, is able to interact with enzyme systems which are critical to important nuclear functions and to interfere--in a cell specific manner--with histones and/or matrix proteins, resulting in greater chromatin compactness.

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and on the DNase I-G actin complex.

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I-globular (G) actin complex were investigated. DNase I, DNase I-G actin complex, and G actin were exposed to various (0.2-4.0 vol%) isoflurane concentrations for 180 min. Thereafter, DNase I activity was determined. DNase I activity was inhibited in relation to time and concentration of isoflurane exposure. At concentrations ranging from 0.2 to 1.0 vol% of isoflurane inactive DNase I was activated in the DNase I-G actin complex. The DNase I inhibitor G actin showed a reduced capability to inhibit DNase I following isoflurane exposure. Albumin can inhibit the DNase I inactivation possibly by competition in the reactions between DNase I/albumin and isoflurane. After exposure to isoflurane the absorption maximum of DNase I was identical with the absorption maximum of heat-denatured DNase I. The results suggest a mechanism by which isoflurane may affect DNA in an indirect way at concentrations to which the patient is exposed during clinical anesthesia.