Alkaline Dissociation Research Articles

Получение изолированных клеток эпидермиса и дермы посредством метода клеточно-щелочной диссоциации

Получение изолированных клеток эпидермиса и дермы посредством метода клеточно-щелочной диссоциации

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L-lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI-MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L-lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer-specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L-lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations.

PH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from − 48.0 mL/mol of subunit at pH 6.0 to − 19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was + 0.602 mol of H +/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein–protein interactions and of the surrounding conditions that modulate the process of aggregation.

Effect of endothelin-1 on apoptosis, proliferation, and protein synthesis in cardiomyocytes of newborn albino rats.

Five intraperitoneal injections of endothelin-1 (100 microg/kg) to newborn albino rats on days 2-6 of life did not change the number of nuclei expressing PCNA in the left-ventricular myocardium. The number of nucleoli, area and perimeter of cardiomyocytes (isolated by the method of alkaline dissociation) increased. The number of myocyte nuclei in the state of apoptosis (evaluated by the TUNEL method) increased significantly. Presumably, partial loss of cardiomyocytes as a result of apoptosis activation after treatment with endothelin-1 is compensated by increased size and transcription activity of remaining cardiomyocytes.

Apoptosis of cardiomyocytes as extreme manifestation of regeneration and plastic insufficiency of myocardium.

Alkaline dissociation of the myocardium from rats with modeled anthracycline cardiomyopathy revealed decreased absolute number of cardiomyocytes, disturbances in their intracellular regeneration, although no sings of necrosis were observed. Regeneration and plastic insufficiency of the myocardium due to structural changes in the nuclei and disturbances in myofibril reproduction resulting from selective suppression of synthesis of contractile proteins in the cardiomyocytes leads to the death of up to 30% cardiomyocytes.

A re-evaluation of the molecular mass of earthworm extracellular hemoglobin from meniscus depletion sedimentation equilibrium. Nature of the 10 S dissociation species

Previous calculations from meniscus depletion sedimentation equilibrium of the molecular mass of earthworm hemoglobin from Lumbricus terrestris (E.J. Wood et al., Biochem. J. 153 (1976) 589–596) and from the related species Lumbricus sp. ( L. sp.) (M.M. David and E. Daniel, J. Mol. Biol. 87 (1974) 89–101) were made on the assumption that the solutions behaved ideally. Re-examination of their results reveals, however, a dependence of the apparent molecular mass on concentration. Taking this effect into consideration, we have now recalculated from their data molecular masses of 4.4–4.5 MDa for the hemoglobin of both L. terrestris and L. sp. On the basis of the new determinations, we propose for the polypeptide chain composition of L. terrestris hemoglobin a model [( abcd) 4L1L2L3] 12 where a,b,c,d are the four globin and L1,L2,L3 are the three major linker chain constituents of the protein. The model is consistent with the D 6 symmetry of the molecule. A 10 S intermediate product in the alkaline dissociation of Lumbricus hemoglobin is viewed as a binary mixture of products resulting from a disproportionation reaction involving the structural unit. The present interpretation is shown to be consistent with observed relations between molecular masses and SDS gel electrophoretic band patterns of 10 S species and intact hemoglobin.

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study.

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

Fluorescence Studies of Extracellular Hemoglobin of Glossoscolex paulistus in Met Form Obtained from Sephadex Gel Filtration

Abstract Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5–8.0 and a second reversible hemichrome is formed above pH 9.5–10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation.

Purification of ferulic acid by adsorption after enzymic release from a sugar-beet pulp extract

An aqueous sugar-beet pulp extract obtained by pressure-cooking and mainly composed of pectic fragments, was treated with a pectinolytic preparation. A strong, but limited depolymerization occurred fastly, while feruloyl-esterase activities appeared more gradually, pointing to some deficiencies of the enzymic mix. The solubility of ferulic acid in the sugar-rich, acidic hydrolysate proved stable, and no crystallization could be triggered in this complex medium. Comparative batch tests were carried out with preselected, commercially-available adsorbents: activated carbons, polystyrenic divinylbenzene-crosslinked resins, and polyvinylpolypyrrolidone (PVPP). In each case, the ferulic acid uptake is lower at pH values above 4.5, the pKa determined for its carboxylic group. The natural acidity of the hydrolysate, pH 3.4, was chosen as the optimum. The chemically-activated carbon (SA1817) has the highest affinity for ferulic acid, while Amberlite XAD-16 performs best among the polystyrenic resins. PVPP also offers an appreciable adsorption efficiency. Selectivity is ensured, since only the charcoal removes a slight amount of galacturonic acid, which is released on rising the pH to 4. Alkaline dissociation and ethanolic extraction were investigated as desorption strategies, and ethanol was kept as the most suitable common eluent. Dynamic column studies allowed to estimate the maximum specific capacity at about 22, 12 and 8% (w:w ferulic acid/adsorbent) for the chemical granular activated carbon (GAC), XAD-16 and PVPP, respectively. Ferulic acid is quantitatively recovered after ethanolic elution of the three selected adsorbents, and the best purity is achieved with PVPP, followed by GAC.

Human pre-alpha-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain.

Pre-alpha-inhibitor (P alpha I) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing P alpha I. Then, taking advantage of the differential affinity of the members of the inter-alpha-inhibitor family (I alpha I) for heparin-Sepharose and blue-Sepharose, we isolated P alpha I. Its specific antitryptic activity was 580 IU/g, higher than that of I alpha I: 420 IU/g. Its M(r), determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130,000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from P alpha I by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the HI and H2 heavy chains of I alpha I. In reducing conditions, it was quite similar to that of H2 (M(r) 85,000) but clearly different from that of H1 (M[r] 78,000). Thus, the so-determined apparent M(r) of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74,100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69,454) are really occupied by two N-glycans, probably of biantennary type.

Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus

Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 × 10 6 Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5–10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal FeN histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0–8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme-globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.

Fluorescence studies of extracellular hemoglobin of Glossoscolex paulistus obtained by gel filtration

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus presents a unique band at pH 7.0 and several bands at pH 9.0 as a result of alkaline dissociation. SDS-polyacrylamide gel electrophoresis (PAGE) of the intact protein obtained at pH 7.0 shows the existence of five different bands with molecular weights of 12 ± 1, 26 ± 3, 28 ± 2, 34 ± 1 and 53 ± 1 kDa. In the presence of β-mercaptoethanol, six distinct bands are obtained with molecular weights 13 ± 1, 14.8 ± 0.3, 15.8 ± 0.2, 16.6 ± 0.2, 35.1 ± 0.3 and 40.5 ± 0.5 kDa. Reduction of disulfide bonds of a trimer of molecular weight 53 kDa leads to the appearance of monomeric subunits of 14.8, 15.8 and 16.6 kDa. The molecular weights obtained from chromatography in Sephadex G-200 at pH 9.0 are different from those from SDS-PAGE. An intense band at 110 ± 12 kDa due to the tertiary complex of two disulfide-linked trimers and monomer chain (trimer + monomer), (abcd)2, is observed in addition to a small amount of high molecular weight fraction in the exclusion volume, as well as poorly resolved trimer (42 ± 2 kDa) and monomer (15 ± 2 kDa) bands. Fluorescence quantum yields of different fractions obtained in the chromatography as well as extinction coefficients at 280 and 415 nm were estimated and compared with human hemoglobin. The fluorescence data presented are consistent with the high content of aromatic residues in the G. paulistus hemoglobin. The increase in the fluorescence quantum yields upon both alkalinization and dissociation were correlated with the exposure of tryptophan residues to the solvent. The intact native protein has a quantum yield of 0.25% at pH 7.0, assigning 14% to tryptophan solution at pH 7.0 and increasing to 0.8% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 0.8% at pH 7.0, increasing to 2.1% at pH 9.0. The maximum emission wavelength of the intact protein (327 nm) is consistent with tryptophan residues buried, quenched and becoming more exposed and more fluorescent upon dissociation into smaller subunits (332-335 nm). In the monomer the tryptophan residues remain buried inside the protein molecule at pH 9.0.

An improved method of mouse liver micronucleus analysis: an application to age-related genetic alteration and polyploidy study

The performance of a micronucleus test in liver cells in vivo requires two laborious procedures: stimulation of hepatocytes to division and dissociation of liver tissue into a single-cell suspension. We propose the method of inhalation treatment of mice with carbon tetrachloride to induce cell proliferation and alkaline dissociation of previously fixed tissue. The micronucleus incidence and ploidy classes in terms of cytophotometric DNA content were determined in liver of mice of three age groups (around 2.5, 5.0 and 7.0 months old) after CCl 4 treatment or partial hepatectomy. The data obtained show that both methods give the same results. The fraction of micronucleated hepatocytes was 0.69% at the age of 2.5 months; it increased to 8.5% and then to 13.5% at 5.0 and 7.0 months respectively. Simultaneously, the ploidy classes changed both with the aging of the animal and after induced liver regeneration. The percentage distribution of micronucleated cells by ploidy class showed that cells carrying micronuclei were the higher ploidies rather than the population in general. Since polyploid cells contain multiple molecular targets for genetic damage, the micronucleation index per genome unit was estimated. Then the real rate of accumulation of both intrinsic endogenous (and probably the exogenously induced) preclastogenic genetic alterations in hepatocytes during the adulthood of mice was evaluated to be 0.03% per diploid genome per day. This seems to be the first description of the phenomenon of liver cell aging in terms of micronuclear aberrations.

Effect of repeated complete starvation on quantitative parameters of rat cardiomyocytes

Sixty-four male Wistar rats were subjected to repeated complete starvation with free access to water. Each starvation period lasted 6 days and was followed by a 7-day recovery period. The concentration of cardiomyocytes (CMC) and their total number in the left ventricle (LV) were measured after alkaline dissociation of the myocardium. The volume density of muscle fibers and their absolute total mass in the LV were determined by stereological analysis. It is shown that after 6 successive starvation periods the proliferative potential of the CMC population is reduced.

A Differential Cloning Procedure of Complex Genomic DNA Fragments

We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (∼10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.

Proteinase-Sensitive Sites on Isolated Rabbit Dystrophin1

Dystrophin was isolated from the purified large oligomeric dystrophin complex with its associated proteins (DC) of rabbit skeletal muscle by alkaline dissociation followed by gel filtration to remove the associated proteins. Isolated dystrophin and DC were subjected to digestion with calpain or alpha-chymotrypsin, and the generated polypeptide fragments were studied by immunoblot analysis using seven kinds of antibodies raised against antigens corresponding to various regions from the N- to the C-terminal of human dystrophin. For some fragments, the amino acid sequences at the N-termini were determined. Two proteinases, which bear distinct specificities, generated very similar fragments from purified dystrophin with or without the associated proteins. The cleavage sites found by mapping the fragments onto the dystrophin molecule were similar to those found in a previous study using crude mouse muscle cell membrane fraction [Koenig, M. & Kunkel, L.M. (1990) J. Biol. Chem. 265, 4560-4566]. On the basis of these results, we concluded that dystrophin has several unique proteinase-sensitive sites.

Purification of dystrophin from skeletal muscle

Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7–12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy < deoxy < CO < met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0–7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.

Disoxaril Stabilization and Immunogenicity of Poliovirus Procapsids

Disoxaril (5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]-heptyl]-3- methylisoxazole) protects poliovirus procapsids against alkaline dissociation and thermal denaturation up to 42 degrees C. When added during the purification of procapsids, it enhances their yield and antigenic quality. Disoxaril increases the immunogenicity of both purified virions and procapsids in mice. Whereas untreated procapsids mainly elicit H-specific antibodies, disoxaril-treated procapsids yield high titres of neutralizing antibodies. The prospect of using disoxaril-treated procapsids in vaccines is discussed.