Reverse hyaluronan substrate gel zymography procedure for the detection of hyaluronidase inhibitors

Little is known of the ubiquitous inhibitors of hyaluronidase, molecules that may be important for the deposition of hyaluronan. A reverse hyaluronan-substrate gel procedure is described here that detects such inhibitors, even in crude biological extracts, and is independent of the catalytic mechanism of the target enzyme. Following electrophoresis, hyaluronan-containing gels are incubated in a hyaluronidase solution. Alcian blue-staining bands indicate hyaluronan protected from degradation and the location of hyaluronidase inhibitors. Coordinated use of hyaluronan substrate gel and reverse substrate gel procedures provides estimates of the number and relative molecular sizes of both enzymes and their inhibitors.     

Affinity capture electrophoresis for sequence-specific DNA purification

A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecules and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.     

Anomalous properties of water in macromolecular gels.

Low molecular weight solutes often exhibit elution characteristics on gel filtration columns which deviate from ideal behaviour. In many previous studies this anomalous behaviour was attributed to the existence of extremely narrow pores in the gel, inaccessible even to very small solute molecules, to explain Kd values lower than unity. Kd values of small solutes higher than unity were usually ascribed to adsorption of the solute to the gel matrix. In the present paper several observations are presented that contradict these suggestions. Experimental evidence indicates that with small solute molecules Kd values differing from unity can be fully explained by the anomalous properties of vicinal water layers at the gel matrix-water interface.     

Target immunity of Mu transposition reflects a differential distribution of Mu B protein

A DNA molecule carrying Mu end DNA sequence(s) is a poor target in the Mu DNA strand-transfer reaction, a phenomenon which is referred to as "target immunity." We find that Mu B protein stimulates intermolecular strand-transfer by binding to the target DNA. Our results show that a differential distribution of Mu B protein between "immune" and "non-immune" DNA molecules is responsible for target immunity; in the presence of Mu A protein and ATP, Mu B protein dissociates preferentially from immune DNA molecules. Hydrolysis of ATP is implicated in establishing the differential distribution of Mu B protein between immune and non-immune DNA molecules in the presence of Mu A protein; nonhydrolyzable ATP gamma S can support an efficient strand-transfer reaction even with a target DNA that is immune in a reaction with ATP.     

Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis.

The uptake and cellular metabolism of a fluorescein-labelled synthetic ribozyme stabilized by 2'- O -methyl modification and a 3' inverted thymidine have been studied, employing capillary gel electrophoresis as a novel and efficient analytical method. After internalization by DOTAP transfection, electrophoretic peaks of intact ribozyme and different degradation products were easily resolved and the amount of intracellular intact ribozyme was quantified to >10(7) molecules/cell at the peak value after 4 h transfection. On further incubation the amount of intracellular intact ribozyme decreased due to both degradation and efflux from the cell. However, even after 48 h incubation there were still >10(6) intact ribozyme molecules/cell. Clear differences both in uptake and in metabolism were seen when comparing DOTAP transfection with the uptake of free ribozyme. Fluorescence microscopy studies indicated that the ribozyme was mainly localized in intracellular granules, probably not accessible to target mRNA. This implies that agents able to release the intact ribozyme from intracellular vesicles into the cytosol should have a considerable potential for increasing the biological effects of synthetic ribozymes.     

A Versatile Composite Gel Column for the Electrophoretic Separation of Ribonucleic Acid Molecules

A composite gel column system containing 2.4% acry I amide, 0.14% N,n¹-methylene bisacrylamide, 0.6% agarose and 15% glycerol as a component has been developed for the electrophoretlc separation of all kinds of ribonucleic acid molecules extracted from eucaryotlc cells. The separations achieved In this composite gel are due to the sieving of RNA molecules of various sizes with similar charge. As a result of the addition of glycerol the polymerization time of the composite gel is extended and the gel has increased mechanical stability. These stable gels can be sliced at room temperature for such procedures as measuring radioactivity or extraction. Their suitability for staining and absorbance scanning is not altered.     

Phase Transitions in Coarse-Grained Lipid Bilayers Containing Cholesterol by Molecular Dynamics Simulations

Coarse-grained simulations of model membranes containing mixtures of phospholipid and cholesterol molecules at different concentrations and temperatures have been performed. A random mixing without tendencies for segregation or formation of domains was observed on spatial scales corresponding to a few thousand lipids and timescales up to several microseconds. The gel-to-liquid crystalline phase transition is successively weakened with increasing amounts of cholesterol without disappearing completely even at a concentration of cholesterol as high as 60%. The phase transition temperature increases slightly depending on the cholesterol concentration. The gel phase system undergoes a transition with increasing amounts of cholesterol from a solid-ordered phase into a liquid-ordered one. In the solid phase, the amplitude of the oscillations in the radial distribution function decays algebraically with a prefactor that goes to zero at the solid-liquid transition.     

Fibrillar nucleolar remnants do not contain macromolecular precursors of ribosomal RNA : Demonstration by the effects of -galactosamine

Administration of -galactosamine to rats produces inhibition of liver nuclear RNA synthesis and associated alterations in the structure of the nucleolus. Polyacrylamide gel electrophoretic analysis of liver nuclear RNA from galactosamine-treated rats has shown the virtual complete absence of ribosomal RNA (rRNA) precursor molecules at a time when the nucleolus consists solely of a dense fibrillar core devoid of granules. No evidence for an artefactual, preferential breakdown of nuclear RNA during extraction could be obtained from either 2.7 or 8% acrylamide gels. Furthermore, the almost complete cessation of nuclear RNA synthesis makes the possibility of there being rapid synthesis and degradation of ribosomal precursor molecules in vivo unlikely. With toluidine blue stains for RNA with nuclei isolated from galactosamine-treated animals, the large, brightly staining area associated with the normal nucleolus was not seen. On the basis of these observations, it is concluded that an RNA-depleted nucleolus appears fibrillar. It is suggested that the fibrillar material of a normal nucleolus may not itself be RNA even though this region does contain RNA precursor molecules.     

Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries.

We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge. A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli. Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones. Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information.     

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.     

A polarized photobleaching study of DNA reorientation in agarose gels

Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that "reversible" photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.     

Target-assembled ExciProbes: application to DNA detection at the level of PCR product and plasmid DNA

Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5'-GCCAAACACAGAATCG-3') was embedded in long DNA molecules (PCR products and approximately 3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% trifluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na(+), K(+), and Mg(2+)) and PCR additives on exciplex emission has been also examined.     

Heat-induced reversible gelation of arachin: kinetics, thermodynamics and protein species involved in the process

Arachin forms a heat-reversible gel under certain experimental conditions. The minimal gelling concentration for this system is 7.25%. Above minimal gelling concentration calculation of thermodynamic parameters for gelation of arachin revealed a constant delta Hbonding (-1220 cal.mol-1) where delta Sbonding values varied with an increase in protein concentration (ranging from -4.01 e.u. at 7.5% to -3.48 e.u. at 10.0%). The main steps involved in the gelation phenomenon include thermal denaturation of arachin, partial aggregation of heat-denatured protein molecules, setting of protein solution and maturation of the gel formed. Gel maturation process follows first order kinetics and is characterized by a large positive delta G+(+) (22,030 cal.mol-1). Determination of delta H+(+) and delta S+(+) for this process revealed that mostly delta S+(+) (-62.9 e.u.) contributes to the large positive delta G+(+), thus decreasing the overall rate of gel maturation process. This large negative delta S+(+) value probably arises from a loss of entropy of protein molecules because of their increased involvement in gel network formation. The polymer gel network seems to be primarily contributed by a part of both arachin dodecameric and hexameric species.     

Nanovector therapeutics

An ideal injected therapeutic drug would travel through the vasculature, reach the intended target at full concentration, and there act selectively on diseased cells and tissues only, without creating undesired side effects. Unfortunately, even the best current therapies fail to attain this ideal behavior, by a wide margin. A primary reason is the fact that the target recognition abilities of the current therapeutics molecules are quite limited. Furthermore, the natural defenses of the body present a sequence of formidable obstacles on the drug's pathway to the intended lesion. Requiring any molecule to have sufficient therapeutic efficacy, target recognition specificity, as well as all of the tools required to bypass multiple biological barriers is probably unrealistic. A different approach is to decouple the problem (i.e. employ the drug molecules for their therapeutic action only, and deliver them to the intended site by vectors that can be preferentially concentrated at desired body locations through the concurrent action of multiple targeting mechanisms). These vectors must also be large enough to comprise all the requirements for the evasion of the body defenses, while still sufficiently small so as not to create undesired blockages of even the smallest of blood vessels - and thus, by definition, nanotechnological.     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Revisiting the mystery of fibronectin multimers: The fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds

Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein–protein bonds.     

Heterogeneity of protein–polysaccharides of porcine articular cartilage. The sequential extraction of chondroitin sulphate–proteins with iso-osmotic neutral sodium acetate

Protein-polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein-polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein-polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein-polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein-polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein-polysaccharides of earlier extracts even though these contained some large molecules.     

Evidence for active half-molecules of alpha 2-macroglobulin formed by dissociation in urea

Urea caused dissociation of alpha 2-macroglobulin (alpha 2M) into half-molecules (two disulfide-bonded subunits) as revealed by gel electrophoresis. The fraction of whole molecules remaining decreased with increasing urea concentration. Half-dissociation occurred at about 2.2 M. The ability of alpha 2M to inhibit trypsin also decreased with increasing urea concentration, but the activity-urea curve was shifted to the right as compared to the dissociation-urea curve. Thus, at 3 M urea, gel electrophoresis showed only 6.6% whole molecules, whereas the trypsin inhibitory activity was 95% of that in buffer with no urea, suggesting that half-molecules retain activity. In addition, complexes formed in urea with 125I-labeled trypsin were observed to migrate as half-molecules even though only 50% of such complexes were covalent. These results are surprising in light of the report by Gonias and Pizzo [Gonias, S., & Pizzo, S. (1983) Biochemistry 22, 536-546] that half-molecules formed by mild reduction are active; reduction is assumed to divide the molecule along an axis orthogonal to the break caused by urea. This suggests that active half-molecules can be formed by splitting either the covalent or noncovalent bonds that hold the subunits together. A model is proposed that can account for this possibility. It has the same dimensions and symmetry as a previous model of Feldman et al. [Feldman, S.R., Gonias, S.L., & Pizzo, S.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704] and accounts in a similar way for previous functional studies of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)