Isopycnic Gradient Centrifugation Research Articles

The effect of the 13C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP.Key points• Appropriate 13C-DNA amount was needed for DNA-SIP.• Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions.• Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.

Optimizing ultracentrifugation conditions for DNA-based stable isotope probing (DNA-SIP)

DNA-SIP (DNA-based stable isotope probing) is increasingly being employed in soil microbial ecology to identify those microbes assimilating the 13C/15N labelled substrate. Isopycnic gradient centrifugation is the primary experimental process for conducting DNA-SIP. However, diverse centrifugal conditions have been used in various recent studies. In order to get the optimum conditions of centrifugation for DNA-SIP, centrifugation time (36, 42, 48, 60 h), speed (45,000, 55,000 rpm) and the initial buoyant density (1.69, 1.71, 1.725 g ml−1), as were used extensively in related studies, were tested in this experiment with the Vti 65.2 rotor. DNA with either 13C-labelling or unlabelled was extracted from a paddy soil pre-incubated with either 13C-labelled or natural abundance glucose. After ultracentrifugation, the gene abundance of bacterial 16S rRNA, fungal 18S rRNA, bacterial and archaeal amoA within the fractioned DNA was detected. The results showed that centrifugation for 48 h was enough for the DNA to reach stabilization in the CsCl solution. The initial density of the mixed solution was best adjusted to 1.71 g ml−1 to ensure that most of the genes were concentrated on the middle fractions of the density gradient. Increasing the centrifugation speed would increase the density gradient of fractions; therefore, 45,000 rpm (184,000 g) was recommended so as to obtain the more widespread pattern of DNA in the centrifugal tube. We hope these findings will assist future researchers to conduct optimum ultracentrifugation for DNA-SIP.

Quantitative Determination of the Carboxyl Groups on Individual Nanoparticles by Acid‐Base Titrimetry

AbstractQuantification of the active groups on nanoparticle surface greatly facilitates the downstream functionalization, which is essential for their application. Among the common active groups, carboxyl group is one of the most popular groups. Hence, we used an improved gravimetric method to obtain the accurate number of the nanoparticles and further determined the number of the carboxyl groups on individual nanoparticles with titrimetry. In this work, polymer nanoparticles with carboxyl groups on their surfaces were taken as a model to validate this determination. Firstly, by using the total weight of the nanoparticles divided by the weight of one nanoparticle, their number concentration was determined. The total weight was obtained by measuring the dry mass of the nanoparticles, and the unit weight was calculated by the volume of a single nanoparticle multiplied with its density, in which the volume was characterized by transmission electron microscopy (TEM), and the density was determined by isopycnic gradient centrifugation. Directly using the real density of nanoparticles achieved more accurate quantification than the current gravimetric method which used the density of their bulk material counterparts for calculation. Then, a back titration was used to determine the carboxyl groups: Certain amount of nanoparticles were first to react with excess NaOH standard solution, and after complete reaction, the residual NaOH were measured by titration with HCl standard solution, from which the amount of the carboxyl groups on individual nanoparticles could be calculated.

A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies.

Recently, a quasi-lipid-enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated. Cell culture lysates and supernatants of two cytopathic HAV variants, HM175/18f and HM175/24a, were characterized for their LE and NLE particle content by isopycnic gradient centrifugation. The obtained fractions were characterized for relative infectivity and then subjected to heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) in 12.5% human serum albumin to investigate their respective heat sensitivity. Preparations of the two HAV variants contained either LE particles (HM175/24a) or LE and NLE particles (HM175/18f) with equivalent specific infectivity. For HM175/18f, heat sensitivity of LE and NLE fractions did not differ significantly, and inactivation of the whole virus stock was identical to the NLE particle inactivation profile, whereas the HM175/24a variant was more heat sensitive. The results indicate that, in heat-treatment studies, the LE or NLE HAV phenotype is less important than the choice of HAV variant, and the most heat-resistant HM175/18f should be used.

Determination of the Absolute Number Concentration of Nanoparticles and the Active Affinity Sites on Their Surfaces.

Number concentration of nanoparticles is a critical and challenging parameter to be identified. Recently, gravimetric strategy is a fundamental method for absolute quantification, which is widely accepted and used by researchers, yet limited by the inaccuracy in measuring related parameters (e.g, density). Hence, we introduced isopycnic gradient centrifugation to determine the nanopartices' density and improved the current gravimetric method for more accuracy. In this work, polymer nanospheres were used as a model to validate this method. Through isopycnic gradient centrifugation, nanospheres finally reached the zone of equal density as them. By measuring the density of the medium solution in this zone, the nanospheres' density was identified. Then, the density was multiplied by the volume of a single nanosphere characterized by transmission electron microscopy (TEM), and the average weight of a single nanosphere was obtained. Using total weight of the nanospheres divided by the unit weight, their number concentration was quantified. Directly using the real density of the nanoparticles achieved more accurate quantification than the current gravimetric method which used the density of the bulk material counterparts for calculation. Besides, compared with the viscosity/light scattering method and the high-sensitivity flow cytometry (HSFCM) method (another two kinds of typical methods respectively based on light measurements and single particle counting), the improved gravimetric method showed better reproducibility and more convenience. Further, we modified the nanospheres with streptavidin (SA) and antibody, and through biorecognition interaction, we determined the amount of the active affinity sites on each biofunctional nanosphere. Moreover, their bioactivity in different storage conditions was monitored, which showed good stability even in PBS at 4 °C over one year. Our work provided a promising method for more accurately determining the absolute number concentration of nanoparticles and the active affinity sites on their surfaces, which would greatly facilitate their downstream applications.

A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation.

Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection.

Problem‐solving test: Transport of a viral protein in a cell‐free system

Terms to be familiar with before you start to solve the test: protein glycosylation, glycoprotein, endoplasmic reticulum, Golgi complex, mannose, oligosaccharide, free polysomes, cell fractionation, [(35) S]methionine; pulse labeling; nuclear fraction, mitochondrial fraction, microsomal fraction, cytosol, gel electrophoresis, autoradiography, trypsin, deoxycholate, detergent, sucrose density gradient, isopycnic gradient centrifugation.

RNA Isolation from Mammalian Samples

RNA can be extracted from cultured cells, peripheral blood, bone marrow, plasma, serum, body fluids, and fresh or frozen tissues. RNA can also be extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Methods for RNA extraction can be divided into three groups: phenol/chloroform extraction, silica spin-column absorption, and isopycnic gradient centrifugation. Two different RNA isolation procedures are described in this unit. The first basic protocol describes a one-step isolation involving monophasic lysis with guanidine isothiocyanate and phenol followed by chloroform extraction. The second basic protocol describes a silica-column separation method for RNA isolation.

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle.

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10−2 pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.

Purification of a herpes-like virus from abalone ( Haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy

A herpes-like virus was for the first time purified from abalone diagnosed with ganglioneuritis. Pleuropedal ganglia, pedal nerve cords, head and epipodial tissue was collected and homogenized from abalone populations exhibiting high mortality and clinical signs consistent with herpes-virus like ganglioneuritis. Following ultracentrifugation by sucrose gradient prepared in sea-water, the purified virus was negatively stained and examined under a transmission electron microscope. Virus particles were observed to have an icosahedral capsid appearance surrounded by an envelope with numerous spikes on the external surface. The capsid ranged 92–109 nm in diameter and the enveloped virus was approximately 150 nm in diameter. Virus particles were found mainly at the interface of 40–50% sucrose gradients, and a few presented at the interface of 50–60% sucrose gradients. Isopycnic gradient centrifugation was performed in a potassium tartrate gradient and caesium chloride gradient, where the buoyant density of the herpes-like virus was determined to be 1.17–1.18 g/mL. The use of sea-water as the buffer in preparation of the gradient was critical in the preliminary purification of the herpes-like virus, and more efficient harvesting of the virus was achieved by sucrose and potassium tartrate gradients than caesium chloride gradient. The described method, whilst proving successful for purifying a herpes-like virus from abalone, may also be applicable to other viruses from marine animals.

Platelet Tetraspanin Complexes and Their Relation to Lipid Rafts.

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. CD63 is located in the membranes of lysosomes and dense granules in resting platelets. Following platelet activation and granule exocytosis, CD63 is expressed on the platelet plasma membrane and co-localizes with the αIIbβ3-CD9 complex. D545, a monoclonal antibody (MoAb) directed at the second extracellular loop of CD63, inhibits activated platelet spreading on immobilized fibrinogen and FAK phosphorylation in the adherent platelets. To identify CD63-associated signaling enzymes that could be involved in the signaling complex, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase with the enzymatic properties of PI4-kinase type II, confirmed by re-precipitation and immunoblotting with 4C5G (MoAb specific for the 55kDa PI4-kinase, PI4K55). The CD63-PI4K55 complex could be co-precipitated from both resting and activated platelets using anti-CD63 MoAb, and co-localized on the filopodia of thrombin-activated platelets using immuno-electron microscopy. Previous studies have demonstrated that tetraspanins associate with cholesterol-enriched membrane domains in a variety of cells including platelets. There is evidence, however, that these tetraspanin-enriched microdomains (TEMs) can be distinguished from prototypic lipid rafts on the basis of detergent solubility and protein composition. To investigate the association of the CD63-PI4K55 complex with lipid rafts in platelets, resting and thrombin-activated platelets were lysed in buffer containing either 1% Brij 35, or Triton X-100, the low- and high-density membrane fractions separated by isopycnic sucrose gradient centrifugation, and the identification of the low-density membrane fractions (LDMF) confirmed by the presence of LAT. CD63, CD9 and PI4K55 were present in the LDMF of platelets lysed in Brij 35 but not in Triton X-100; they were also present in the denser membrane fractions. CD63 and CD9 associated with cholesterol, as demonstrated by recovery of these proteins in the pellet following centrifugation of platelets lysed with 1% digitonin(a cholesterol-precipitating reagent), but not from lysates made with Brij 35/Triton X-100. Incubation of platelets with methyl-β-cyclodextrin(mβCD) to partially deplete cholesterol and disrupt the lipid rafts shifted LAT, CD63, CD9 and PI4K55 to denser fractions within the gradient. Immunoprecipitation of mβCD-treated platelets with anti-PI4K55 MoAb co-precipitated CD63 and CD9, demonstrating that the complexes were not dependent on residence within LDMFs, but remained intact in the denser fractions and pellet. Platelet tetraspanin complexes associate with cholesterol-enriched domains under conditions of mild detergent extraction. The maintenance of the complexes, however, was not dependent on their residence within lipid rafts, as the complexes remained intact following cholesterol depletion. Their presence in LDMF suggests that tetraspanin complexes may associate with platelet lipid rafts under some conditions, which could bring tetraspanin protein partners into proximity with raft residents, and facilitate the assembly and interaction of signaling complexes following platelet activation.

Impacts of viability and purification on the specific gravity of Cryptosporidium oocysts

The specific gravity of unpurified and purified Cryptosporidium oocysts was measured using an isopycnic gradient centrifugation technique. Specific gravity varied depending on the viability of the oocysts, as defined by permeabilty to 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), the presence or absence of internal structures, and whether or not oocysts were purified. The modal range of densities for a population of 1.4-week-old unpurified oocysts, was 1070–1073 kg/m 3. This range was higher than that determined for 14-week-old purified oocysts, 1067–1070 kg/m 3. Eleven- and 12-week-old unpurified populations exhibited a bimodal distribution of densities, with densities most frequently in the 1005–1041 and in the 1077–1108 kg/m 3 range. In these populations, a high percentage of the oocysts having densities greater than 1077 kg/m 3 were viable, while oocysts in the 1005–1024 kg/m 3 range were predominately nonintact, and oocysts in the 1024–1041 kg/m 3 range were intact, but permeable to DAPI and PI (nonviable). This work demonstrates the importance of controlling factors that may impact the viability of oocysts when conducting studies that examine the transport of these microorganisms in the environment and through water treatment processes.

Protein kinases and their protein substrates in free messenger ribonucleoprotein particles and polysomes from mouse plasmacytoma cells.

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)-cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle-associated cAMP-independent protein kinase(s) using [gamma-32P]ATP and [gamma-32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP-independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.

Purification of Enterocytozoon bieneusi from Stools and Production of Specific Antibodies

Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 microm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.

Identification of alternatively spliced human biotinidase mRNAs and putative localization of endogenous biotinidase

Biotinidase is essential for recycling the vitamin biotin and for transferring biotin to proteins, such as histones, suggesting that the enzyme localizes to various cellular and extracellular sites. To better understand the functions of the enzyme, we examined its gene structure and subcellular localization. Using RACE-PCR and a BLAST search, we extended the 5 ′ sequence of the biotinidase gene. Three novel, alternatively spliced variants of biotinidase, 1a, 1b, and 1c, were identified in multiple human tissues. Exon 1c is present only in testes. The sequence of the 5 ′ splice variants, 1a and 1b, suggest that biotinidase localizes to the mitochondria and/or ER, respectively. Using indirect immunofluorescence studies, biotinidase localizes to organelles in the cytoplasm, but not nucleus, of human fibroblasts and Hep G2 cells. Endogenous expression was examined by isopycnic gradient centrifugation of rat liver organelles, which identified an 85 kDa biotinidase protein with biotinyl-hydrolase and transferase activities in microsomes and possibly lysosomes. A 48 kDa protein, which also reacts with anti-biotinidase, localizes to mitochondria. The 48 kDa protein is not N-glycosylated but is biotinylated, is in the inner mitochondrial matrix, but has no biotinyl-hydrolase or transferase activities. The function and validation of the mitochondrial species remains to be determined. The 5 ′ splice variants and organelle fractionation studies indicate that biotinidase is directed to the secretory pathway and perhaps mitochondria.

Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells.

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

In situ localization and in vitro induction of plant COPI-coated vesicles.

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.

Relative contributions of mouse liver subcellular fractions to the bioactivation of mitomycin C at various pH levels

Mitomycin C (MMC) is a clinically active anticancer drug that requires reductive activation to exert its toxicity. The enzymes currently recognized as capable of activating MMC cannot account for all of the toxicity of the drug. These studies were conducted to identify and compare the subcellular compartments where MMC reduction can take place under different physiological conditions. Subcellular fractionation of mouse liver was achieved using differential centrifugation and isopycnic equilibrium gradient centrifugation. Nuclear, mitochondrial, microsomal, lysosomal, peroxisomal, and cytosolic fractions were assayed for their ability to reductively activate MMC at pH 6.0 and 7.4. MMC reductive activation was determined by its ability to generate reactive oxygen species. The results of these studies showed that MMC reductive activation by the various fractions was pH dependent. At pH 7.4, the microsomal fraction accounted for approximately 78% of the total MMC reductive activation. The peroxisomal fraction accounted for 12% and the nuclear and lysosomal fractions each accounted for 5% of the total reductive activation. At pH 6.0, the microsomes accounted for 51% and the peroxisomes for 34% of the total reductive activation. The mitochondrial fraction, which did not reductively activate MMC at pH 7.4, accounted for 9% of the total activation at pH 6.0. These results suggested that peroxisomes may be important in MMC activation at either pH and that at pH 6.0 the mitochondrial fraction may also be important for MMC reductive activation.

A 110-kDa Nuclear Shuttle Protein, Nucleolin, Specifically Binds to Adeno-Associated Virus Type 2 (AAV-2) Capsid

A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed thein vitrobinding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin bothin vitroandin vivosuggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly.

Isolation and characterization of the outer membrane of Borrelia hermsii.

The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable beta-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.