Isopycnic Sedimentation Research Articles

Liver Sinusoidal Endothelial Cell (Lsec) Isolation Following a Liver Perfusion Technique

Introduction: Liver perfusion has been the standard method to digest and isolate liver cells including liver sinusoidal endothelial cells (LSEC). Poor cannulating skills through portal vein results in a waste of animal resource. Familiarization of both liver perfusion technique and adhering strictly to aseptic technique during cell handling ensure high cell yield, minimum morphology disruption and cell contamination. We aimed to present a method of liver perfusion procedure followed by the isolation of LSEC. Materials and method: The study was conducted with the approval of IACUC committee. Seven Sprague Dawley rats underwent these procedures under anaesthesia. Liver perfusion was done as previously described. Briefly, LSEC were isolated by liberase enzyme perfusion of the liver, isopycnic sedimentation in a two- step Percoll gradient and selective adherence. The purification and cultivation of LSEC was evaluated by light and electron microscopy. Results: Purity and viability of LSEC after selective adherence was 80.5 ± 3.5% and ≥ 95 %, respectively. The average concentration of the cells ranged from 32 - 75 x 106 per 400 gm rat. After 8 hours of culture, LSEC monolayers were contaminated with less than 5% of other cells. Conclusion: This method is reliable and reproducible for the isolation of LSEC to enable the study of structure and function of these cells in vitro. However, improvement on the perfusion skills and isolation technique are vital to ensure better cell purity.

Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes.

The majority of porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%). They therefore are probably free molecules in solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-to-minus-strand molar ratio of approximately 8:1 and viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs.

Nucleotide sequence and protein analysis of a complex piscine retrovirus, walleye dermal sarcoma virus

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3' end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue. Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.

Isolation and Characterization of the Small Nucleolar Ribonucleoprotein Particle snR30 from Saccharomyces cerevisiae

The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1. All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation. NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the small nucleolar ribonucleoprotein particle snR30 (snoRNP). In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein. Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure. The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients. Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide.

Simultaneous purification of chloroplast and mitochondrial DNA without isolation of intact organelles from green carrot tissue and suspension cultures

We report here the simultaneous purification of chloroplast (cpDNA) and mitochondrial DNA (mtDNA) from green tissue and suspension cultures of carrot without organelle isolation. This method is based on isolating total nucleic acids from frozen tissue and separating the nuclear, chloroplast and mitochondrial fractions using sequential isopycnic sedimentation in two gradients of cesium chloride containing bisbenzamide. From 10 g of mature carrot leaves, 10 to 30μg of organelle DNA was consistently recovered from mature carrot leaves, while 30 to 50 μg was recovered from suspension cells. The method can be used to isolate chloroplast and mitochondrial DNAs from single plants without sacrificing the individual.

Localization of Chitin Synthase inAbsidia glaucaStudied by Immunoelectron Microscopy: Application of Cryoultramicrotomy

Application of cryoultramicrotomy to immunoelectron microscopy showed the localization of the chitin synthase of Absidia glauca in situ by using anti-chitin synthase IgG. Mild fixation with a mixture of 0.25% glutaraldehyde and 4% formaldehyde did not destroy the antibody-binding capacity of chitin synthase and preserved the ultrastructure of organelles. Infusion with a mixture of polyvinylpyrrolidone and sucrose as a cryoprotectant before freezing provided adequate sectioning plasticity. Immunolabeling of colloidal gold was detected both at the plasma membrane and the vesicles, and more so in the latter. The results of immunoelectron microscopy were consistent with the fractionation profile of isopycnic sedimentation of the microsomal fraction on a linear sucrose gradient. The profile also demonstrated two peaks of chitin synthase, one of these cosedimented with vanadate-sensitive ATPase and the other with lower buoyant density populations. The former corresponded to the plasma membrane and the later, ve...

Luminal and basolateral uptake and degradation of insulin in the proximal tubules of the dog kidney.

In order to determine the major routes of insulin degradation in the body, insulin was labelled with a 'trapped' or 'residualizing' label: [125I]tyramine-cellobiose ([125I]TC) and injected intravenously in dogs. In contrast to conventional iodine-labelled insulin (131I-insulin), the [125I]TC-insulin allows measurements of total uptake in specific organs in vivo because the radioactive degradation products do not leave the cells. One h after the injection of trace doses, the amount of radioactivity recovered in the kidney from [125I]TC-insulin was nine times higher than when conventional [131I]insulin was used. In the blood, the amount of acid-precipitable radioactivity was the same for both labelled preparations, indicating similar clearance rates. A comparison of the uptake of insulin in filtering vs. non-filtering (ureter-occluded) kidneys indicated that the uptake of insulin is twice as high through the luminal than through the basolateral cell membrane; after 60 min, 8.9 +/- 0.8% of the injected [125I]TC-insulin dose remained in the filtering kidney and 3.2 +/- 0.2% of the dose was accumulated in the contralateral kidney, with occluded ureter but normal blood perfusion. In both filtering and non-filtering (ureter-occluded) kidneys, the subcellular distributions of [125I]TC-insulin were studied after various times by isopycnic sedimentation in sucrose gradients. No difference between peritubular and tubular uptake was discernible. The intracellular transport was rapid, leading to accumulation of radioactive label in dense lysosomes within 10 min.

Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells.

The studies that document the coupling of strand-specific DNA repair to transcription of active genes exclude replicated DNA from the analysis. Yet cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light (UV) persist in most of the genome in surviving Chinese hamster ovary (CHO) cells. The mechanisms that allow DNA replication to occur in the presence of damaged templates are poorly understood. We have investigated the distribution of CPD in the dihydrofolate reductase gene (DHFR) domain in replicated DNA. CHO B11 cells were incubated in the presence of BrdUrd after UV irradiation; the replicated DNA was separated from the unreplicated DNA by isopycnic sedimentation in CsCl, and then the parental and daughter strands were resolved in alkaline CsCl. We determined the fraction of a 14-kb KpnI fragment of the DHFR gene that was resistant to digestion by T4 endonuclease V, a CPD-specific enzyme. In both parental and unreplicated DNA, approximately 80% of the CPD were removed from the transcribed strands while approximately 20% were removed from the nontranscribed strands of DHFR within 24 h. In a 15-kb KpnI fragment that contains an origin of replication and is located approximately 15 kb downstream of DHFR, we found very low repair levels, whether it had been replicated or not. We detected no CPD in the daughter strands of either fragment analyzed. These results suggest that the replication forks can move through the damaged DNA in the absence of significant levels of repair or strand exchange and that the repair of CPD is not affected by replication in these cells.

Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification

Because of their intrinsic low buoyant density, chitosomes can be separated from crude cell homogenates (1000 g or 35,000 g supernatants) of Mucor rouxii by isopycnic sedimentation in sucrose density gradients. To accelerate and simplify the isolation of chitosomes, we centrifuged the cell-free extracts at ultrahigh speed (in a fixed-angle rotor at forces up to 311,000 g Rav) and found that the duration of centrifugation was critical. Prolonged centrifugation at ultrahigh speed caused severe distortion of the chitin synthetase profile in the gradient as the peak of chitosomal chitin synthetase nearly disappeared. We traced the problem to a soluble protease(s) that moved into the chitosome band during protracted centrifugation and destroyed the chitin synthetase activity. The interfering protease was a soluble protein with a sedimentation coefficient of 4.6 S and a pH optimum of 7-7.5, and it was sensitive to PMSF (phenylmethylsulfonyl fluoride), indicating that it was a serine protease. Unlike other proteases, it destroyed chitin synthetase but did not activate the chitin synthetase zymogen. The interfering protease could be eliminated either by adding PMSF to the cells immediately after breakage or by removing the upper part of the sucrose gradient midway through the centrifugation of the cell-free extract and then completing the sedimentation with the 'decapitated' gradient.

Sedimentation for the separation of cells

Among the many approaches to the separation of cells, sedimentation either at unit gravity or with a centrifugal force has been widely used. Since the quality of the suspensions of cells prior to sedimentation greatly influences the separation, it is important to evaluate several methods for the dispersion of solid tissues to obtain suspensions of viable, single cells. The optimal approach to a particular tissue varies as a function of organ, donor species and age, and other experimental variables. There are two kinds of sedimentation that can be used for cell separation in continuous gradients: velocity sedimentation and isopycnic sedimentation. Isopycnic sedimentation accomplishes cell separations according to the different densities of cells. There are four commonly used forms of velocity sedimentation: sedimentation at unit gravity, isokinetic sedimentation, sedimentation in a reorienting zonal rotor, and elutriation. The choice of method for the separation of particular kinds of cells will depend on the needs of the investigator, including the number of cells desired, the cost, and the need for work under sterile conditions.

Isolation of proteoglycans using phenol extraction and isopycnic centrifugation in cesium trifluoroacetate

A procedure for the rapid isolation of heparan sulfate proteoglycans from Zwittergent lysates of cultured cells and cetylpyridinium precipitates of culture medium is described. After removal of most nonglycosylated proteins by phenol extraction, the proteoglycans are fractionated and isolated by isopycnic sedimentation in cesium trifluoroacetate gradients. The densityisolated proteoglycans, which are largely free of high molecular weight DNA, RNA, and nonglycosylated proteins, are readily amenable to chemical and enzymatic characterization. These procedures, which are designed for efficient and rapid isolation of lipophilic proteoglycans, also appear applicable to the isolation of other high density proteoglycans.

Fractionation of Latex-Based Adhesives and Paints by Isopycnic Centrifugation

Abstract Latex-based adhesives and paints can be fractionated by isopycnic sedimentation in density gradients, generated by osmocentrifugation. Fractions were obtained from a PVA-based adhesive, which differed in tacticity, as observed by 1H NMR; calcium carbonate was easily identified in another product, after fractionation. In an acrylic-based latex paint the pigments contained PVA, although this polymer was not detected in other fractions. These results show that low-speed isopycnic sedimentation in density gradients is a convenient, gentle, inexpensive, and powerful method to fractionate complex particle admixtures. A marked advantage of this method is its ability to give fractions large enough for examination by other analytical techniques.

The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.

A genetically engineered cell line that produces empty capsids of B19 (human) parvovirus.

B19 parvovirus is pathogenic in humans, causing the common childhood exanthem fifth disease and bone-marrow failure, both acute (transient aplastic crisis of hemolysis) and chronic (pure erythrocyte aplasia in immunodeficiency). The virus is tropic for a human red cell progenitor cell, and failure to culture B19 in a cell line has limited its clinical study. We cotransfected the right half of the cloned B19 genome and a minigene derived from the human dihydrofolate reductase gene (DHFR) into dhfr--Chinese hamster ovary cells and screened selected clones by RNA analysis; after amplification in methotrexate, clones were tested for capsid protein expression. A cell line, designated 3-11-5, stably expressed nearly full-length transcripts for the two capsid proteins. These cells produced the major and minor structural protein species in natural proportions that self-assembled into virion capsids. Capsids from 3-11-5 cells could be separated from virions by sucrose gradient sedimentation and had the density on cesium chloride isopycnic sedimentation of empty parvovirus capsids. Capsid protein was present in both nuclei and cytoplasm on immunofluorescence study but fractionated with the cytosol on purification. Empty capsid production was equal to or greater than virion production by infected bone-marrow cells, 1000-2000 capsids per cell, but cell growth was not diminished by capsid production. This cell line will be useful in developing practical assays for B19 parvovirus antibody and a vaccine for the virus, as well as potentially serving as a packaging cell line for gene therapy.

Chromoplast-Specific Proteins in Capsicum annuum

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M(r) of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.

The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component

Gene cpxA of Escherichia coli K12 encodes the 52,000 M r CpxA polypeptide. The complete cpxA nucleotide sequence, reported here, predicted that CpxA contains two extended, hydrophobic segments in its amino-terminal half and could therefore be a membrane protein. Using a lac- cpxA operon fusion plasmid to overproduce CpxA and an immunochemical assay to detect the polypeptide, we show that CpxA fractionated with the bacterial inner membrane during differential and isopycnic sedimentation. Moreover, the protein could be solubilized by extraction of crude membranes with non-ionic detergents but not with KCl or NaOH, indicating that Cpx is an intrinsic membrane component. Analysis of Tn phoA insertions in cpxA indicated that the region between the hydrophobic segments of CpxA is periplasmic, whereas the region carboxy-terminal to the second such segment is cytoplasmic. Based on these structural data, we propose that CpxA functions as a trans-membrane sensory protein. The DNA sequence data also indicate that cpxA is the 3′ gene of an operon.

Comparison of Phosphorus Dynamics in Two Oklahoma Reservoirs and a Natural Lake Varying in Abiogenic Turbidity

The rate of phosphate uptake, the immediate fate of phosphate, and the potential rate of release of phosphate from dissolved phosphorus compounds in epilimnia of two reservoirs and one natural lake in northcentral Oklahoma were compared in the summer and in the late autumn. Sangre Isle Lake is a highly productive eutrophic impoundment, Sanborn Lake is a natural lake with low phytoplanktonic productivity, and Boomer Lake is an impoundment with modest productivity that is heavily affected by clay suspensoids. Although the uptake of phosphate (radiometrically determined) was rapid in each of these lakes in summer, the fate of phosphate in Boomer Lake differed from that of the others, with much of the phosphate sorbing to suspended silts and clays rather than biota (determined by isopycnic sedimentation). Recycling of phosphate from phosphomonoesters was greatest in Sangre Isle Lake, moderate in Sanborn Lake, but was undetectable in Boomer Lake. These findings suggest that high concentrations of clay suspensoids influence the epilimnetic P dynamics in reservoirs and lakes.

Progesterone and testosterone production by dispersed rat placental cells.

Isopycnic separation and unit gravity sedimentation were employed to identify the rat placental cell types capable of producing progesterone and testosterone. Subdivision of Day 12-dispersed placental cells in Percoll gradients revealed that fractions (less than 1.048 g/ml) containing giant cytotrophoblast cells produced greater quantities of progesterone (p less than 0.01) than did fractions (greater than 1.048 g/ml) with equal numbers of placental cells but void of giant cytotrophoblasts. Unit gravity sedimentation of Day 16-dispersed placental cells revealed that when incubated, isolated giant cytotrophoblast cells were capable of producing both progesterone and testosterone. Both of the separation studies strongly suggested that other cell types also produce steroids. However, the biosynthetic capacity of the giant cytotrophoblast cell appeared to be 1000-fold greater than that of the other cell types. Incubation of Day 12-dispersed placental cells with human chorionic gonadotropin or 3',5'-cyclic adenosine monophosphate did not further increase progesterone production as compared to untreated control incubates, suggesting rat placental steroidogenesis is not under trophic hormone control. Electron microscopic observations of giant cytotrophoblast cells revealed a complex ultrastructure suggesting a variety of physiological functions.

The Coat Proteins and Nucleic Acids of Two Beet Cryptic Viruses

Summary Purified particles of beet cryptic virus (BCV) contained two protein species with estimated mol. wt. of 5.45 × 104 and 5.25 × 104, and four nucleic acid species of mol. wt. 1.36, 1.15, 0.94 and 0.87, all × 106. These nucleic acids were shown to be dsRNA on the basis of thermal denaturation profile, isopycnic sedimentation in Cs2SO4 solutions, electron microscopical appearance and nuclease resistance. Previous work has shown that a cryptic virus from leaf beet in Japan reacts with BCV antiserum but contains only two dsRNA species that co-migrate with the two larger RNAs of BCV. These and our results suggest that BCV is a mixture of two viruses, although this was not evident from the appearance of the particles in the electron microscope.

Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4.

The denV gene of phage T4, encoding the pyrimidine dimer-specific DNA repair enzyme endonuclease V, has been introduced by DNA transfection into the UV-sensitive DNA repair-deficient Chinese hamster ovary (CHO) cell line UV5. Transformants were first selected for resistance to the antibiotic G418 conferred by the neo gene from Tn5 carried by the same plasmid. A majority of the isolated G418-resistant UV5 clones also showed an increased resistance to 254-nm UV light. One clone, designated I-A1, was found to have an intermediate level of colony-forming ability after UV irradiation when compared to UV5 and wild-type AA8 cells. A Southern blot showed that I-A1 carries a single integrated intact copy of the denV gene. Alkaline sucrose gradients revealed a dose-dependent appearance of breaks in the DNA of I-A1 cells following UV-irradiation, while unirradiated cells did not exhibit any significant breaks. Analysis of DNA repair by isopycnic sedimentation showed that DNA excision repair by I-A1 was at least equal to the level of repair in AA8 cells. These results show that the prokaryotic denV gene can restore UV repair capabilities in vivo to CHO UV5 cells defective in repair of UV-induced damage.