CsCl Equilibrium Density Gradient Research Articles

Optimized Method for Pseudomonas aeruginosa Integrative Filamentous Bacteriophage Propagation.

Filamentous bacteriophages frequently infect Pseudomonas aeruginosa and alter its phenotypic traits, including virulence factors. The first step in examination of these phages is to obtain suspensions with high virus titer, but as there are no methods for integrative filamentous phage multiplication, the aim was to design, describe, and compare two methods for this purpose. As models, three strains of Pseudomonas aeruginosa, containing (pro)phages Pf4, Pf5, and PfLES were used (PAO1, UCBPP-PA14, and LESB58, respectively). Method 1 comprised propagation of phages in 6 L of bacterial culture for 48 h, and method 2 applied 600 mL culture and incubation for 6 days with centrifugation and addition of new medium and inoculum at 2-day intervals. In method 1, phages were propagated by culture agitation, followed by centrifugation and filtration (0.45 and 0.22 μm), and in method 2, cultures were agitated and centrifuged several times to remove bacteria without filtration. Regardless of the propagation method, supernatants were subjected to concentration by PEG8000 and CsCl equilibrium density gradient centrifugation, and phage bands were removed after ultracentrifugation and dialyzed. In the obtained suspensions, phage titer was determined, and concentration of isolated ssDNA from virions was measured. When propagation method 2 was compared with method 1, the phage bands in CsCl were much thicker, phage number was 3.5–7.4 logs greater, and concentration of ssDNA was 7.6–22.4 times higher. When phage count was monitored from days 2 to 6, virion numbers increased for 1.8–5.6 logs, depending on phage. We also observed that filamentous phage plaques faded after 8 h of incubation when the double layer agar spot method was applied, whereas the plaques were visible for 24 h on single-layer agar. Finally, for the first time, we confirmed existence of replicative form and virions of PfLES (pro)phage as well as its ability to produce plaques. Similarly, for the first time, we confirmed plaque production of Pf5 (pro)phage present in P. aeruginosa strain UCBPP-PA14. The described method 2 has many advantages and can be further improved and adopted for filamentous phages of other hosts.

Perennial Prescience.

Perennial Prescience.

Large scale purification of RNA nanoparticles by preparative ultracentrifugation.

Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

Human Adenoviruses: Propagation, Purification, Quantification, and Storage

Detailed protocols are described for the propagation of adenoviruses (Ads) and adenovirus (Ad) vectors and their purification by CsCl equilibrium density gradient centrifugation. A discussion of monolayer and spinner cell culture techniques suitable, respectively, for small- and large-scale growth of adenoviruses is provided. Protocols for cloning into and growth of Ad replication-deficient vectors using a convenient commercially available system are described. Lastly, time-tested plaque titration protocols for the accurate and convenient measurement of the infectivity of adenoviruses and adenovirus vectors are provided in detail.

Hydroxyapatite Chromatography of Phage-Display Virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.

Circulating autoantibodies against purified colonic mucin in ulcerative colitis

To clarify the role of colonic mucin in the autoimmune process of ulcerative colitis, circulating antibodies against human colonic mucin were investigated. Purified colonic mucin, obtained from human colonic mucosa by gel filtration, using a Bio-Gel A-1.5-m column and CsCl equilibrium density gradient, was divided into soluble mucin (S-mucin) secreted extracellularly and membranous mucin (M-mucin) binding to cell membrane. Sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting analysis showed that antibodies in the serum samples of some patients with ulcerative colitis recognized purified S- and M-mucin of >180-kD. By enzyme-linked immunosorbent assay (ELISA), anti-mucin antibodies were detected in 11 of 60 patients with ulcerative colitis (18%). In contrast, the antibodies were not detected in 22 patients with Crohn's disease. The titers of antimucin antibodies against S-mucin and M-mucin were not different in each patient. By ELISA using mucin in which the sugar chains were destroyed by neuraminidase or NaIO4 treatment, it was demonstrated that anti-mucin antibodies recognized the epitopes of either the sugar chain or the core protein exposed through destruction of the sugar chains. We then investigated the relationship between anti-mucin antibodies and the patients' clinical features. Anti-mucin antibodies were detected in 6 of 15 patients with chronic continuous type ulcerative colitis (40%) and in 5 of 26 patients with relapsing-remitting type (19%), but there was no antimucin antibody-positive serum in patients who had had only one attack without any relapse. These results suggest that anti-mucin antibodies could be a disease marker for ulcerative colitis and that immunological abnormalities in colonic mucin contribute to the persistence of colonic mucosal inflammation.

The maize mitochondrial plasmid RNA b is associated with protein during synthesis but is not encapsidated.

RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in maize mitochondria. The extent to which this molecule is associated with proteins was investigated by rate zonal and CsCl equilibrium density gradient centrifugation of clarified lysates of S cytoplasm maize mitochondria. A soluble complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in mitochondrial lysates, was identified. The complex had a buoyant density of 1.49 g/cm3, indicating a substantial non-nucleic acids content. The sedimentation coefficient of the complex, however, was only slightly larger than that of deproteinized RNA b. Synthesis of RNA b as well as the larger RNA plasmid, RNA a, was resistant to heparin, suggesting that, for both RNAs, preformed complexes between an RNA template and an RNA-dependent RNA polymerase capable of elongating in vivo preinitiated RNA plasmid strands, were present in the lysate. Only a small fraction of RNA b molecules were bound in the complex; the bulk of RNA b sedimented at the same rate as the deproteinized RNA. Thus, after replication, maize mitochondrial plasmids are not associated with nucleoprotein capsids although their synthesis takes place through ribonucleoprotein replication complexes.

Inhibition of Protein Synthesis by the Heme‐Controlled Eif‐2α kinase Leads to the Appearance of mRNA‐Containing 48S Complexes that Contain eIF‐4E but Lack Methionyl‐tRNAf

Phosphorylation of the initiation factor eIF-2 by the heme-regulated eIF-2 alpha kinase (HCR) results in pronounced inhibition of protein synthesis and of binding of Met-tRNA(f) to 40S subunits in reticulocyte lysates. This inhibition is associated with the appearance of a more rapidly sedimenting 48S complex; this contains mRNA detectable by poly(U) hybridization, but not Met-tRNA(f). In contrast, 48S complexes that accumulate in the presence of the initiation inhibitor edeine contain both Met-tRNA(f) and mRNA. We have compared the composition of the particles that accumulate in the presence of HCR with those seen in the presence of edeine and find that both particles contain the cap binding protein, eIF-4E. Moreover, both particles exhibit a buoyant density of 1.40 g/ml in CsCl equilibrium density gradients. This is consistent with the presence of 500-700 kDa of protein additional to ribosomal structural protein, and suggests the presence of eIF-3 on both types of 48S complex. Lysates pre-treated with HCR and then treated with edeine show the ability to accumulate 48S complexes containing Met-tRNA(f), though at a slower rate than control lysates. These observations are discussed in the light of mechanisms previously suggested for the appearance of 48S particles following HCR treatment. In addition, we have observed association of eIF-4E with polysomes and 80S monosomes in reticulocyte lysates, suggesting that this factor may not be released immediately following the binding of the 40S ribosomal subunit to the 5' end of the mRNA.

Preliminary characterization of mucin from effusions of cleft palate patients

Middle ear effusions from children undergoing myringotomy were classified into three groups-cleft palate, thick (mucoid), and thin (serous). Mucin was purified from each of the three groups using CsCI equilibrium density gradient centrifugation. Analysis of the cleft palate mucin on Sepharose CL-2B showed it was excluded and therefore of large molecular weight. It could be broken down into smaller glycopeptide units by proteolysis and these glycopeptides had, based on elution position, a larger hydrodynamic size than those from the thick mucin. Intrinsic viscosity measurements demonstrated that the intact mucins could be ranked in order of molecular space occupancy; cleft palate > thick > thin. Amino acid analysis showed the cleft palate mucin to have an amino acid composition similar to other mucins, with serine, threonine, and proline constituting 41% by weight of the protein core. Thiol analysis gave evidence of a possible difference in polymerization between the three mucins, in that thin (the smallest mucin) contained the lowest number of thiols. This preliminary analysis of cleft palate mucin suggests a mucin with larger glycopeptide units forming an intact mucin of larger hydrodynamic size than either thick or thin middle ear mucins from anatomically normal children.

The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica.

The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.

Inhibition of Chondroitin Sulfate Incorporation into Human Thyroglobulin byp-Nitrophenyl-β-D-Xylopyranoside*

Human thyroglobulin (TG) is unique among glycoproteins and TGs of other species in having a chondroitin sulfate chain. We studied the effects of p-nitrophenol-beta-D-xylopyranoside (PNXP), an inhibitor of chondroitin sulfate incorporation into core protein of proteoglycans, on the synthesis of human TG. Fragments of normal thyroid tissue from two patients were preincubated for 1 h with PNXP before adding [35S] sulfate and [3H]leucine. TG was purified by ammonium sulfate precipitation, sucrose gradient centrifugation, and CsCl equilibrium density gradient centrifugation. Chondroitin ABC lyase released 38-42% of the [35S]sulfate from control TG, synthesized in the absence of inhibitor, demonstrating the presence of chondroitin sulfate units. In the samples incubated with increasing concentrations of PNXP (0.2 and 1 mM), the fraction of [35S] sulfate released by ABC lyase decreased progressively (14-23% and 8-11%, respectively). This confirms that, as in chondroitin sulfate proteoglycans, chondroitin sulfate synthesis in TG is initiated by the transfer of a galactose unit, by galactosyltransferase, to a xylosyl-serine in the TG peptide backbone. This step is prevented by PNXP, which is a competitive acceptor of galactose. The density of TG synthesized in the presence of PNXP was determined by CsCl equilibrium density centrifugation. [3H] leucine-labeled TG from the samples incubated with PNXP was less dense than [3H]leucine-labeled TG from the control specimen. The newly synthesized TG was less dense either because of the lack of the chondroitin sulfate chain or because further processing of the TG was impaired. To differentiate these possibilities, we treated control, [3H]leucine-labeled TG with chondroitin ABC lyase and compared its density to that of [3H] leucine-labeled TG synthesized in the presence of 1 mM PNXP. The decrease in density produced by PNXP was greater than the decrease brought about by chondroitin ABC lyase (4.3-5.2 x 10(-3) and 2.4-2.8 x 10(-3) g/cm3, respectively). Therefore, the density contributed by the chondroitin chain was insufficient to account for the entire density shift seen with PNXP. We conclude that 1) chondroitin sulfate synthesis is initiated by the transfer of a galactosyl unit to xylosyl-serine in the TG peptide backbone; and 2) inhibition of this step decreases the density of TG by preventing the addition of the chondroitin chain and interfering with the further processing of TG.

Effect of ethanol on the in vitro sulfation of salivary mucin.

In vitro sulfation of mucus glycoprotein by sulfotransferase from rat submandibular salivary gland and the effect of ethanol on this enzyme activity was investigated. Subcellular fractionation studies revealed that the enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein is associated with Golgi-rich membrane fraction. The sulfotransferase enzyme exhibited optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 4 mM MgCl2, and 25 mM NaF. The enzyme was equally capable of sulfation of the desulfated intact as well as proteolytically degraded desulfated glycoprotein preparations, whereas the acceptor capacity of the intact mucus glycoprotein was 70% lower. The submandibular gland sulfotransferase activity was inhibited by ethanol. The rate of inhibition of mucus glycoprotein sulfation was proportional to the concentration of ethanol up to 0.4 M, at which concentration a 39% reduction in the sulfotransferase activity occurred. The apparent Km value of the enzyme for salivary mucus glycoprotein was 11.1 microM, and the Kl in the presence of ethanol was 0.93 M. The synthesized 35S-labeled glycoprotein gave on CsCl equilibrium density gradient centrifugation 35S-labeled peak which coincided with that of the glycoprotein. Alkaline borohydride treatment of this glycoprotein led to the liberation of the label into the acidic oligosaccharide alditol fraction. As the inhibition by ethanol of sulfotransferase enzyme occurred below its isosmotic concentration to plasma, the observed effect could also be detrimental to salivary mucus glycoprotein sulfation in vivo.

Initial Characterization of small Proteoglycans Synthesized by Embryonic Chick Leg Muscle-Associated Connective Tissues

Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.

Induction of cellular DNA synthesis in G 0-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses

tsJT60 cells, a temperature-sensitive G 0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 °C) and nonpermissive (39.5 °C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G 0 phase) they entered S phase at 34 °C but not at 39.5 °C. Infection of G 0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant d1313, and Adl2 wild type and its E1B mutants in205B, in205C, d1205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.

Single-strand displacement replication of bacteriophage M2 DNA.

Replicative intermediates of M2 DNA, pulse-labeled with [3H]-thymidine, were isolated and the mode of its replication was examined. Compared with mature M2 DNA the replicative intermediates had the following properties: i) higher sedimentation coefficient in sucrose gradient, ii) higher buoyant density in CsCl equilibrium density gradient, and iii) sensitivity to nuclease Sl. Two types of intermediates were found by electron microscopic observation. One is unit-length duplex molecules with a single-stranded branch of various lengths. The other is unit-length linear molecules consisting of one single-stranded and one double- stranded region of variable proportions. Distribution of the radioactivity in pulse-labeled completed molecules was analyzed with restriction endonucleases. Both ends of the genome had higher radioactivity, showing that the ends are termini of replication. These results indicate that the replication of M2 DNA proceeds from each end by displacing parental single-stranded DNA.

Double-stranded DNA of soybean chlorotic mottle virus.

The nucleic acid of soybean chlorotic mottle virus (SoyCMV) was isolated and analysed. CsCl equilibrium density gradient centrifugation and thermal denaturation kinetics revealed that the nucleic acid is a double-stranded DNA. Electrophoresis in agarose gels separated SoyCMV DNA into multiple bands, similar to those of cauliflower mosaic virus (CaMV) DNA, which indicated that this DNA consisted of both linear and circular molecules. The existence of these different topological forms was confirmed by electron microscopy. Using six different restriction endonucleases a physical map was constructed which showed the SoyCMV genome to be a circular DNA molecule of approximately 8, 200 base pairs long. The results obtained support the previous assignment of SoyCMV to the caulimovirus group.

The plastome of a brown alga,Dictyota dichotoma

Plastids of the brown algaDictyota dichotoma contain a single homogeneous DNA species which bands at a buoyant density of 1.693 g/cm(3) in neutral CsCl equilibrium density gradients. The corresponding nuclear DNA has a density of 1.715 g/cm(3). The molecular size of the plastid DNA is 123 kbp as calculated by both electron microscopy of spread intact circular molecules and gel electrophoresis following single and double digestions with various restriction enzymes. A restriction map has been constructed using the endonucleases Sal I, Bam HI, and Bgl II which cleave theDictyota plastome into 6, 12, and 17 fragments, respectively. No large repeated regions, as found in chlorophycean andEuglena plastid DNAs, were detected.Dictyota dichotoma is the first member from the chlorophyll c-line of the algal pedigree for which a physical map of plastid DNA has been established.

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [ 3H]palmitic acid and [ 3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [ 3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [ 3H]palmitate and [ 3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/ palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.

Purification method for the avian adeno-associated virus

Avian adeno-associated virus (AAAV) pre-purified by Uvasol extraction, one discontinuous and two CsCl equilibrium density gradients, was still considerably contaminated with avian adenovirus (CELO strain). Four different approaches were investigated in attempts to improve the elimination of the contaminating CELO virus. The contaminations were assayed by double immunodiffusion and indirect immunofluorescence. The immunoprecipitation of CELO virus with antiserum and protein A-Sepharose is the most effective method of obtaining purified AAAV free of CELO virus.

Purification and hemagglutinating properties of egg drop syndrome 1976 virus.

We purified three populations of virus particles, F7, F9 and F17, with buoyant densities of 1.34, 1.33 and 1.29 g/ml, respectively, in CsCl equilibrium density gradients from cultures of chick embryo liver cells infected with the H-162 strain of the virus of egg drop syndrome 1976. F9 particles were infectious complete virions and most F17 particles were empty particles. F7 particles were less infectious, and had little capacity of hemagglutination (HA). HA titers were the same at 4 degrees and 37 degrees C and maximal between pH 6.4 and 8.4 and ionic strength from 0.14 to 0.54 M of NaCl. HA titer was inversely proportional to erythrocyte concentration. Potassium periodate destroyed markedly the infectivity of the virus and partially its HA activity at 37 degrees C. HA activity was stable at 56 degrees C or lower temperatures and destroyed at 80 degrees C. Trypsin, alpha-chymotrypsin, papain, ficin and neuraminidase had no effect on HA activity. Alpha-chymotrypsin destroyed the receptor for the virus on chicken erythrocytes, whereas trypsin and neuraminidase did not affect the receptor.