Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics.

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.

Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial.

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.

Effects of autologous, cross-linked erythrocytes on isolated hypoperfused rabbit heart dynamics.

The present study was aimed at assessing the effects of either red blood cells (RBC) or RBC cross-linked with the bifunctional dimethyl suberimidate reagent (C-RBC) on contractile force (CFo), heart rate (HR) and coronary flow (CF) of the isolated rabbit heart hypoperfused with RBC suspensions under 30 mm Hg constant pressure. RBC or C-RBC caused a rapid and marked reduction of CF, CFo and HR. In RBC-treated hearts, however, reperfusion with Tyrode solution partially restored the initial myocardial parameters, while in C-RBC-treated hearts a rapid impairment of diastolic relaxation with a subsequent, steady and increasing heart contracture was observed. Histological analysis showed that in C-RBC-perfused hearts either capillaries or precapillary arterioles were occluded by C-RBC in spite of extensive washings with Tyrode solution. These findings indicate that C-RBC impair coronary circulation markedly and irreversibly.

Properties of the 120,000- and 95,000-dalton actin-binding proteins from Dictyostelium discoideum and their possible functions in assembling the cytoplasmic matrix.

The cell cortex of Dictyostelium amebae contains an actin-rich cytoplasmic matrix. Changes in geometry of this matrix are believed to regulate protrusive activity and motility of the cell cortex. Two actin-binding proteins (120,000 and 95,000 daltons [120K and 95K]) are present in the cell cortex, and their properties, many of which are described here for the first time, suggest that they regulate growth and organization of cortical microfilaments. The 120K protein is a flexible dimer 35 nm in length with a native molecular mass of 241,000. It nucleates the polymerization of actin and crosslinks the filaments to form branched networks like those seen in situ in the cell cortex. The production of a branched network of short crosslinked filaments results in a lattice that would theoretically generate the maximum rigidity with minimum amount of polymer. This sort of lattice would be very useful as a space-filling cytoskeleton capable of resisting deformation. The 120K protein inhibits the actin-stimulated Mg ATPase of myosin. Competition for actin binding between 120K and myosin, the impenetrability of the 120K-actin network to myosin, and the rigidity of actin filaments that are crosslinked by 120K could all contribute to the decrease in the actin-stimulated Mg ATPase of myosin. The properties of 120K are consistent with a role for this protein in regulating the site of actin filament growth and gelation in the cell but not the assembly of actin-containing structures that would participate in force generation by a sliding-filament mechanism involving myosin. The 95K protein is a rigid dimer 40 nm in length with a native molecular mass of between 190,000 and 210,000. Its physical and antigenic properties lead us to conclude that the 95K protein is Dictyostelium alpha-actinin. Unlike 120K, it crosslinks actin filaments into lateral arrays and increases the actin-stimulated Mg ATPase of myosin. Both activities are regulated by Ca2+. The properties of 95K are consistent with a role in organizing actin filaments in the cell into lateral arrays that are capable of efficient interaction with myosin to produce force for cell motility.

Fibrous apatite grown on modified collagen.

Fibrous apatite has been grown by the enzymatic hydrolysis of calcium beta-glycerophosphate on reconstituted calfskin collagen tapes which had been modified by the addition of a phosphoprotein, phosvitin, in the presence of a cross-linking agent, dimethylsuberimidate. The deposits were identified as a carbonate-bearing hydroxyapatite by x-ray diffraction, and scanning electron micrographs confirmed their fibrous character.

Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry.

This report describes the fluorescence hybridization of DNA sequence probes to interphase nuclei in suspension and the quantification of bound probe by dual beam flow cytometry. Nuclear proteins were first cross-linked with dimethylsuberimidate to prevent disintegration of the nuclei during denaturation and hybridization. To demonstrate that in situ hybridization can be performed in suspension, stabilized mouse thymocyte nuclei were hybridized with a probe for mouse satellite DNA sequences. The DNA probes were labeled with 2-acetylaminofluorene. After hybridization, an indirect immunofluorescent labeling procedure was used to visualize the target sequences. With dual beam flow cytometry, both the amount of hybridized probe and the DNA content of individual nuclei were determined. Thus, the specificity of DNA hybridization can be combined with the speed and quantitative analysis provided by flow cytometry.

[Inhibition of functional activity of macrophages in vitro by dimer RNase Bacillus intermedius].

The influence of cross-linked by dimethylsuberimidate dimeric RNAse from Bacillus intermedius on peritoneal rat macrophages has been investigated in vitro. It has been shown that dimeric RNase with concentrations of 0.5-40.0 mg/ml decreases the functional activities of macrophages. This is manifested in the inhibition of the phagocyte function of macrophages and suppression of the fusion of phagosomes with lysosomes. The change in the cytoplasmatic membrane surface structure induced by the dimers, which is stronger than that induced by monomers, has been demonstrated using atomic force microscopy. The role of membrane properties modification in the inhibition effect of RNase dimers on the functional activities of macrophages is discussed.

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degrees C.

Structure of bacterial fatty acid synthetase from Brevibacterium ammoniagenes.

Hydrodynamic measurements and a cross-linking study with dimethyl suberimidate have shown that the native fatty acid synthetase from Brevibacterium ammoniagenes is a hexameric protein having a molecular weight of 1.56 . 10(6). The subunits of the enzyme are identical in size (Mr 2.6 . 10(5). The negatively stained fatty acid synthetase had an electron microscopic image of ellipsoidal structure with major and minor axes approximately equal to 270 A and 180 A, respectively. The electron microscopic image is similar to that of the yeast enzyme, which is quite distinct from the B. ammoniagenes enzyme with respect to the subunit composition. The inactivated enzyme prepared by dialysis against a lower ionic strength solution was partially reactivated by raising the ionic strength. Ellipsoidal images similar to those of the native enzyme were found in the electron micrograph of the reactivated enzyme. Sucrose density gradient centrifugation of the reactivated enzyme sample showed that the active component had almost the same sedimentation coefficient as the native hexamer. These results indicate that the enzyme is active only in its hexameric state.

Lipid arrangement in fluid model membranes: analysis by cross-linking of phosphatidylethanolamines.

Binary mixtures of fluid phase phosphatidylethanolamines at pH 10 were treated with the bifunctional cross-linking reagent dimethylsuberimidate. Analysis of the dimeric species formed demonstrated that the phospholipid species in dimyristoylphosphatidylethanolamine/dielaidoylphosphatidylethanolamine mixtures at 52 degrees C and dielaidoylphosphatidylethanolamine/dilauroylphosphatidylethanolamine mixtures at 41 degrees C were randomly arranged. Analysis of the dimeric species formed in dipalmitoylphosphatidylethanolamine/dioleoylphosphatidylethanolamine mixtures at 68 degrees C showed that this mixture was very close to being randomly arranged, with just a slight propensity of like phospholipid species to cluster.

The role of crotoxin subunits in tropical rattlesnake neurotoxic action.

The major toxin (crotoxin) of Crotalus durissus terrificus (neotropical rattlesnake) is known to be a reversible non-covalently associated complex consisting of an acidic and basic subunit. On separation biological activity is found only with the basic subunit, yet, although void of detectable biological activity, the acidic subunit is essential for the full neurotoxic activity of the complex. Recent evidence suggests that crotoxin A serves as a 'chaperone' to enhance the specificity of crotoxin B and, upon binding, crotoxin A is released to the medium. This study was designed to test this hypothesis. Dimethyl suberimidate, a bifunctional cross-linking agent, was used to irreversibly bind the two subunits. Disc electrophoresis, ion-exchange chromatography, molecular sieve chromatography, capillary isotachophoresis and isoelectric precipitation confirm the existence of an inter-subunit covalently cross-linked complex. The conversion of a dissociable complex to a non-dissociable complex abolished neurotoxicity. Although neurotoxicity was lost, phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), which is found associated with many presynaptic neurotoxins, was unaffected. The data in this paper add credence to the 'chaperone' concept of crotoxin A and the importance of the reversible nature of the complex for full expression of neurotoxicity.

Two different forms of aggregated dimers of ribonuclease A.

Results of gel filtration experiments performed with two different chromatographic media (Superose 12 HR 10/30 and Superdex 75 HR 10/30) and of polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions suggest that aggregated dimers of bovine RNase A, obtained by lyophilization of the enzyme from 40% acetic acid solutions (5 mg RNase A per ml), might consist of two differently structured forms. These two species have slightly different retention times in gel-filtration experiments and migrate differently in electrophoresis under non-denaturing conditions. The fast migrating dimer in non-denaturing gel electrophoresis is able to degrade double-stranded poly(A).poly(U) more efficiently than the other, and the two forms are found in a ratio of about 3:1.

Cross-linking of phosphatidylethanolamine neighbors with dimethylsuberimidate is sensitive to the lipid phase.

Dimethylsuberimidate was reacted with aqueous dispersions of dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, and dielaidoylphosphatidylethanolamine at pH 10 and at pH 8. The amount of amidine dimer formation was about four times greater above the gel-to-fluid phase transition of each lipid than below the transition. The transition temperature of each phosphatidylethanolamine, measured by steady-state fluorescence anisotropy of cis-parinaric acid, was lower at pH 10 than at pH 8 or in water. The ability of dimethylsuberimidate to discriminate between phosphatidylethanolamines in the fluid and gel phases should allow use of this reagent to identify phosphatidylethanolamine species within the gel or fluid lipid phase.

Characterization of subspecies from a fungal fatty acid synthetase.

Fatty acid synthetase from the filamentous fungus, Aspergillus fumigatus dissociates during gel filtration on Sepharose 6B into two differently sized subspecies with mol. wt. approx. 1.5 x 10(6) and 8 x 10(5). After elution, they readily reform intact molecules, as determined by their enzymic activity (overall synthetase and 3-oxoreductase activities were measured), sedimentation coefficient and appearance in the electron microscope. Synthetase was cross-linked with dimethyl suberimidate and the resultant protein did not dissociate on Sepharose 6B. The two smaller species which were eluted after chromatography of untreated enzyme were also fixed by reaction with this reagent. They did not reform intact molecules of synthetase and were characterized by electron microscopy as large and small circular aggregates; the low molecular weight form also contained tetrameric structures which exhibited cyclic symmetry. The composition of the two species derived during dissociation was, therefore, confirmed as eight and four polypeptides, respectively; each contained polypeptides A and B. It is proposed that the intact fungal synthetase of composition A6B6 comprises three equivalent loops of protein, each of which contain four polypeptides, presumably with composition A2B2; the molecular weights of A and B are 207 000 and 201 000, respectively. During filtration on Sepharose 6B, two such loops remain associated to form a large circle, leaving the other four polypeptides ro rearrange themselves into a small circle or tetramer.

Ca2+-induced conformational changes in the troponin complex detected by crosslinking.

In an attempt to detect Ca2+-induced conformational changes by crosslinking, rabbit muscle troponin complex was reacted with the bifunctional reagents 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone and dimethyl suberimidate under various conditions and the products were analyzed by dodecyl sulfate gel electrophoresis. In the absence of divalent cations, with the two aromatic reagents at a low reagent/protein ratio, the main cross-link products were troponin T-I and I-C. With dimethyl suberimidate the only major crosslink product was conjugate T-I. Ca2+, alone as well as in the presence of Mg2+, prevented the formation of I-C crosslinks with both aromatic reagents, but it did not affect crosslinking with dimethyl suberimidate. Ca2+ also decreased the number of NH2 groups of troponin that are highly reactive towards 2,4,6-trinitrobenzene sulfonate. Both effects of Ca2+ can be interpreted in terms of a conformational change in the troponin complex elicited by the binding of the specific divalent cation.

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. I. Effect of size.

Aspects of protein structure determining endocytosis of proteins by sinusoidal rat liver cells in vivo have been studied, using cross-linked or aggregated derivatives of bovine pancreatic ribonuclease A (labelled with 125I) as probes. Ribonuclease was cross-linked by reaction with dimethylsuberimidate, a way of modification that does not change the charge of the protein. Monomer, dimer and polymer fractions were isolated by gel filtration and characterized in respect of size and number of amino groups modified. Maintenance of enzyme activity, stability of disulfide bonds, and lack of susceptibility to endoproteases showed that the cross-linking procedure did not result in gross conformational changes of the ribonuclease molecules. Monomer, dimer and polymer fractions were injected into nephrectomized rats and plasma clearance and uptake in liver and spleen were determined. About 30% of the injected polymer fraction was found in liver 15 min after injection; for dimer and monomer fractions values of 6% and 2% of the dose were found. Similar differences were found in spleen. Autoradiography, cell isolation, and subcellular fractionation showed that in liver the radioactive proteins were taken up in lysosomes of sinusoidal cells. Similar results were obtained with fractions of aggregated ribonuclease prepared by freeze-drying the protein from 50% acetic acid. Our results demonstrate that the rate of uptake of the ribonuclease derivatives is positively correlated with the size of the molecules. Similarity of the results obtained with cross-linked and aggregated fractions suggests that the number of ribonuclease 'subunits'/molecule, rather than the procedures used to prepare the polymers, determine the rate of uptake by liver and spleen.

Hybrid association between human apolipoproteins A-I and A-II in aqueous solution and in phospholipid recombinants.

The formation of hybrid association products between apolipoprotein A-I and apolipoprotein A-II from human high-density lipoprotein was investigated in solutions of these apolipoprotein and in recombinant particles with dimyristoylphosphatidylcholine (DMPC). It was found that these two proteins interact in solution to form hybrid association products, but not to a marked degree. When these two proteins were incubated together with DMPC, it was likewise found that there was little tendency to reside on the same particle, as judged from the absence of hybrid oligomers by chemical cross-linking. By a modified immunoelectrophoretic method it was found that only about 15% of the A-II and 10% of the A-I were precipitated by the heterologous antiserum; from this it is concluded that 80-90% of these proteins do not form hybrid recombinants with the other protein. These results suggest that in the delipidated state, as well as in discoidal recombinants, there do not exist strong protein-protein interactions between A-I and A-II. This implies that even in the high-density lipoprotein, where both proteins coexist in the same particle, the A-II does not stabilize the molecular structure through interactions with A-I, and its role in this molecule remains obscure.

Investigation of the properties of bovine heart creatine kinase cross-linked with dimethyl suberimidate.

Dimeric bovine heart creatine kinase (EC 2.7.3.2, ATP: creatine N-phosphotransferase) has been cross-linked with the bifunctional reagent dimethyl suberimidate at several concentrations to yield modified enzyme with enhanced stability towards heat denaturation. The degree of thermal stability is dependent on the degree of cross-linking with optimal stabilization occurring when approx. half of all the available amino groups are covalently attached to dimethyl suberimidate. Accelerated storage studies were performed and the results used to predict the storage time of the native and modified enzyme at lower temperatures. The cross-linked derivative was predicted to have a longer shelf-life at 4 degrees C than the native enzyme. Modification caused a reduction in the specific activity of the enzyme. The pH profile was altered following cross-linking, but the Michaelis constants were not changed. The modified enzyme exhibited a marked resistance to the action of some denaturing agents.

Organization of the transcortin-binding domain on placental plasma membranes.

Complex formation between transcortin (corticosteroid-binding globulin) and 20 kDa sialoglycoprotein from human syncytiotrophoblast plasma membranes (presumably a transcortin-recognizing subunit of the transcortin membrane receptor) was studied using FPLC and cross-linking with bifunctional reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of the purified 20 kDa sialoglycoprotein and transcortin resulted in formation of covalently linked complexes of 95 kDa and 140 kDa consisting of one transcortin molecule and either two or four molecules of the membrane sialoglycoprotein (the molecular mass of transcortin is 55 kDa). Additionally, cross-linking resulted in the appearance of a 43 kDa species which is the cross-linked dimer of the membrane protein. The dimer was also observed during chromatography on a Superose 12 column in the absence of DFDNB treatment. Treatment of intact syncytiotrophoblast membranes with DFDNB resulted in isolation of the transcortin binding protein dimer as the major portion of total pool of the protein. Formation of the transcortin complexes with two and four molecules of the membrane protein was also observed when the membranes were incubated with 125I-labeled transcortin and treated with DFDNB, but formation of the latter complexes predominated. The results obtained suggest that the recognizing and binding domain for transcortin in placental membranes is organized as dimers consisting of non-covalently linked sialoglycoprotein monomers of a 20 kDa each and that transcortin has two sites for interaction with this dimer. Apparently, binding of two dimers results in the formation of the functional form of the transcortin-receptor complex. The possible biological role of such a complex is discussed.

Effect of size and charge on endocytosis of lysozyme derivatives by sinusoidal rat liver cells in vivo.

Hen egg-white lysozyme has been modified by intermolecular cross-linking with dimethyl suberimidate or by acylation with acetic or succinic anhydride. Retention of the native conformation of the modified enzyme was checked by measuring enzyme activity, resistance of disulfide bridges to reduction by thiols, and susceptibility to proteases. Unmodified lysozyme and its derivatives (labelled with 125I) were intravenously injected into nephrectomized rats, and plasma clearance and uptake by liver cells were determined. Under these conditions, about 6% of the unmodified lysozyme was taken up by liver 15 min after injection. Cross-linking led to a greatly increased uptake (up to 89% of the dose in 15 min), whereas acylation reduced the uptake to 3-4%. Cell isolations showed that the unmodified enzyme and the cross-linked derivatives were taken up by sinusoidal cells. Differential fractionation of liver homogenates indicated tht the unmodified enzyme was taken up in lysosomes. The cross-linked derivatives were concentrated in the nuclear and microsomal fractions as well as in the lysosomal fraction, suggesting adsorption on plasma membranes besides uptake in lysosomes. The experiments described in this paper, together with previous results on ribonuclease and lactate dehydrogenase, indicate that endocytosis of some proteins by sinusoidal liver cells is positively correlated with size and positive charge of the molecules.