Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.     

Beet curly top virus from phloem exudate of Amsinckia douglasiana

Amsinckia douglasiana when infected with beet curly top virus (BCTV) produces more exudate, which is highly infective, than any other known host. Attempts were made to purify BCTV from the phloem exudate of infected Amsinckia douglasiana by differential centrifugation and sucrose density gradient fractionation. A_260/280 ratio of virus preparations was 1.58; S values were 74 and 147. Infectivity was distributed among several fractions during density gradient purification. Electron microscopy revealed the presence of pentagonal and hexagonal particles of 14–23 nm, some of which were paired. The virus was insensitive to DNAse, RNAse, and trypsin when these enzymes were tested individually but infectivity was substantially decreased when the virus was subjected to trypsin treatment followed by DNAse, suggesting that DNA is associated with the beet curly top virus.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

Partial purification and evidence for multiple molecular forms of the scrapie agent.

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and detergent treatment with sodium deoxycholate yielded a fraction designated "P5" which was enriched for scrapie infectivity approximately 20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction P5 to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from 1.08 to 1.30 g/cm3. Parallel rate-zonal analysis showed that the infectivity was distributed over a range of particle sizes with s20.w values from approximately 40 S to greater than 500 S. Incubation of P5 at 37 or 80 degrees C, under conditions that disrupt ribosomes, dramatically altered the rate-zonal gradient profile of the agent. Under these conditions, the agent sedimented as particles with s20.w greater than 500 S. The apparent heterogeneity of the scrapie agent with respect to both size and density and its ability to shift from one form to another suggest that the agent may contain hydrophobic domains on its surface.     

Attempts at Multiplication,Purification, Electron Microscopy,and Characterisation of Three Isolates of the Strawberry Mottle Agent

Three isolates of strawberry mottle agent (SMA) from strawberry plants were regularly maintained and multiplied by mechanical inoculation onChenopodium quinoa plants showing mosaic and mottle symptoms. The use of 5 mM borate buffer pH 8.6 or tap water pH 6.6-7.9 with 4 % (m/v) charcoal for homogenization resulted usually in 100 % infection. The total of 2090 plants were infected from 2264 inoculated ones under the same conditions. The infectivity of SMA isolates in crude sap ofC. quinoa was retained from 48 h to 72 h at 20 °C. The dilution end points of SMA isolates were 10^-3 while the inactivation temperatures were between 50 and 55 °C. The infectivity of SMA isolates in frozen leaves ofC. quinoa was detected still after six months. Purification procedure of SMA is based on using low molar 25 mM borate buffer pH 8.3 with cysteine hydrochloride, DIECA and Tween 20 for homogenization of infectedC. quinoa leaves, polyethyleneglycol precipitation, clarification with octanol, low and high speed centrifugation and sucrose density-gradient centrifugation. Partially purified preparations are highly infectious, causing mosaic, mottling and tip necrosis ofC. quinoa plants. The agent could not be completely separated from host proteins and it could not be concentrated to a high extent. Isometric virus-like particles 14-16 nm were observed in partially purified preparations.     

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed.     

Zonal Centrifuge Applied to the Purification of Herpesvirus in the Lucké Frog Kidney Tumor

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.     

蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯度分析及与层析法的比较

通过对蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯化工艺进行分析,发现现行工艺中收取的病毒组分中,不同蔗糖浓度中病毒的纯度是不同的,而呈峰型且与病毒效力峰及蛋白浓度峰不重合。实验结果对今后工艺的改进具有指导意义。另外应用Sephacryl-s-1000凝胶层析法对乙脑病毒进行了纯化研究和分析,发现蔗糖梯度离心法和层析法纯化的病毒的纯度及回收率分别为291．46u／mg、96．01％和309．41 u／mg 65．08％,Sephacryl-s-1000凝胶层析法同样可以获得较高的纯度和收率。     

Cellular distribution of 3-hydroxy-3-methylglutaryl coenzyme a reductase and mevalonate kinase in leaves of Nepeta cataria

In Nepeta cataria leaf tissue there are two separate activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and mevalonate (MVA) kinase respectively as determined by the use of a 20–45% discontinuous sucrose density gradient. Cell-free extracts of leaf and callus tissue were prepared and HMG-CoA reductase and MVA kinase activities were compared to activities in extracts from porcine livers and yeast autolysates. Callus tissue from N. cataria has only one peak of HMG-CoA reductase and MVA kinase activity located at the top of the sucrose density gradient. Isolated chloroplast from N. cataria leaves have one peak of HMG-CoA reductase and MVA kinase activity, located near the bottom of a sucrose density gradient. MVA kinase activities in porcine livers and yeast autolysate also showed only one activity profile, located at the top of the sucrose gradient. Partial purification of the leaf extract through the use of differential centrifugation, 30–70% ammonium sulfate precipitation and Bio-Gel P-100 column chromatography shows that MVA kinase, 5-phosphomevalonate (MVAP) kinase and 5-pyrophosphomevalonate (MVAPP) decarboxylase activities remain in the same fractions. The extra-chloroplastidic HMG-CoA reductase activity may be separated from MVA kinase activity by differential centrifugation. These results suggest the presence of two HMG-CoA reductase and MVA kinase enzymes in N. cataria leaf tissue—one located in the chloroplast and a second being extra-chloroplastidic.     

Purification and Characterization of Banana Bunchy Top Virus

Abstract Banana bunchy top virus (BBTV) was successfully purified by a procedure which included pulverizing diseased tissues frozen in liquid nitrogen, clarification of the extract with chloroformbutanol, concentration of the virus by cycles of differential centrifugation, stirring and incubating at 4°C followed by a second cycle of differential centrifugation and sucrose density gradient centrifugation. The concentrations of BBTV obtained from leaf blades and midribs plus petioles were up to 0.34 and 0.56mg/kg tissue, respectively. The virus concentration was highest in diseased leaves collected from October to December, and lowest in those collected from June to September. BBTV is a luteovirus (particles 20– 22 nm in diameter), preparations of which have maximum absorbance at 257 nm, minimum absorbance at 240 nm and a A260/280 ratio of 1.46. The relative molecular mass (M_r) of BBTV coat protein subunit is 21,000, and that of its ssRNA is 2.0 × 10⁶.     

The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein

The cardiac receptor for calcium channel blockers was purified from bovine microsomal membranes which contained 235 +/- 33 fmol nimodipine-binding sites/mg protein (mean +/- SEM of nine preparations). To identify the receptor during the purification 20% of its binding sites were prelabeled with (+)[3H]PN200-110. The receptor was solubilized with 0.6% digitonin and was purified to a specific density of 157 pmol/mg using a combination of ion-exchange, wheat-germ-agglutinin-Sepharose chromatography and sucrose density gradient centrifugation. In the last sucrose gradient bound (+)[3H]PN200-110 comigrated with a 195-kDa protein. ( +/-)[3H]Azidopine and [3H]ludopamil, the photoaffinity ligands for the dihydropyridine and phenylalkylamine-binding site of the calcium channel, were incorporated specifically into the 195-kDa protein. These data indicate that the bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Its molecular mass suggests that the bovine cardiac receptor differs considerably from the rabbit skeletal muscle receptor protein for calcium channel blockers.     

Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation

Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol-dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5'-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.     

Subcellular distribution of the transmissible agent in Creutzfeldt-Jakob disease mouse brain

To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.     

区带超速离心提纯流感病毒的初步探讨

在制备灭活流感疫苗中,采用两次蔗糖速率区带超离心从流感病毒感染的鸡胚尿液中提纯流感病毒是较简便、实用的方法,根据流感病毒分子特性设计了超离梯度并采用适当试验条件,获得了大量提纯流感病毒的初步结果。经血凝、电镜、蔗糖浓度、浮力密度等检验证明此方法是可行的。     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

Serological studies of Bratislava mosaic virus of grape vine

Bratislava mosaic virus was transmitted mechanically toChenopodium quinoa. The virus was isolated by means of gradient ultracentrifugation and the, proteins were distributed on an automatic recording photometer. Single fractions were taken with a fraction collector and used as antigen. At the same time, antigen from leaves of the variety Sylván zelený infected with the Bratislava mosaic virus was prepared by means of the gradient ultracentrifugation. The antisera were obtained from rabbits immunized with individual antigens. The antisera were tested with the saps ofC. quinoa infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Ouchterlony, and with adjusted saps from suckers infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Oudin. A specific reaction can be obtained in both cases only under the assumption that all conditions mentioned in the study are strictly kept.     

Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.     

Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different

We have purified putative L-type Ca2+ channels from chick heart by virtue of their associated high affinity receptors for the Ca2+ channel effectors, dihydropyridines (DHPs), and phenylalkylamines (PAAs). A peptide of 185,000-190,000 daltons was found to comigrate with the peak of DHP binding activity during purification through two successive cycles of lectin affinity chromatography and sucrose density gradient centrifugation. A previously described peptide of 140,000 daltons, whose Mr was increased to approximately 180,000 under nonreducing conditions, also copurified with the 185-kDa peptide and dihydropyridine binding activity. When cardiac membranes were photolabeled with either the dihydropyridine [3H]azidopine or the PAA [3H]azidopamil prior to purification, a single, specifically labeled component of 185,000-190,000 daltons was present in the purified fractions. The properties of this 185-kDa cardiac DHP/PAA receptor were compared to the smaller 165-kDa DHP/PAA receptor previously purified from skeletal muscle. Antibodies raised against the 165-kDa skeletal muscle DHP/PAA receptor reacted with both rabbit and chick skeletal muscle receptors, but only poorly recognized, if at all, the cardiac 185-190 kDa component. The 185-kDa peptide present in the purified fractions obtained from cardiac muscle did not undergo substantial phosphorylation by cAMP-dependent protein kinase, while the purified 165-kDa peptide from rabbit and chick skeletal muscle was a good substrate for this kinase. The results show that the DHP and PAA receptors in cardiac muscle are contained in a 185-190-kDa peptide that is significantly larger than, and structurally and immunologically different from, it skeletal muscle counterpart.     

Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Two procedures for the purification of infectious lymphocytic choriomeningitis virus from cell culture fluid have been developed. If large quantities of very pure virus are to be prepared, infected L cells are maintained with a medium supplemented with calf serum, the proteins of which have been largely removed by pretreatment with polyethylene glycol. Two days after infection of the cultures, the media are collected and the virus is concentrated by treatment with polyethylene glycol 40,000. Purification with a 10,000-fold increase of specific infectivity is achieved with steric chromatography on controlled-pore glass beads with pore sizes of 42 to 44 nm and centrifugation in density gradients prepared with amido trizoate. An alternative method begins with precipitation of the virus from infected cell cuture medium with zinc acetate, followed by controlled-pore glass chromatography and density centrifugation in a discontinuous sucrose gradient. Purification thus obtained is 200-fold in terms of specific infectivity.