CsCl Density Gradient Centrifugation Research Articles

Assembly of Hepatitis Delta Virus Particles: Package of Multimeric Hepatitis Delta Virus Genomic RNA and Role of Phosphorylation

We previously demonstrated that both casein kinase II (CKII) and protein kinase C (PKC) positively modulate the hepatitis delta virus (HDV) RNA replication but not the assembly of the empty hepatitis delta antigen (HDAg) particle. In this study, we investigated whether phosphorylation of HDAg by these two kinases plays a role in assembly of the HDV virion. As demonstrated byin vivolabeling and kinase inhibitor experiments, the phosphorylation level of large HDAg but not small HDAg in HDAg-expressing HuH-7 cells was diminished by CKII inhibitor (DRB), whereas no effect was observed for the phosphorylation level of two HDAgs when treated with protein kinase A (PKA) inhibitor (HA1004) or PKC inhibitor (H7). Cotransfection experiment also demonstrated that packaging of HDV genomic RNA was not affected by the kinase inhibitor DRB or H7 and mutation at the putative CKII phosphorylation sites (serine-2, serine-123, or both), and the putative PKC site (serine-210) of HDAg did not elicit any significant effect on the HDV virion assembly. Therefore, based on the previous work and the present study, it seems that the status and biological significance of phosphorylation of HDAg vary depending on the HDV life cycle. Although in the HDV RNA replication cycle, phosphorylation of small HDAg by CKII or PKC plays important role in HDV replication, phosphorylation of the same HDAg by these two kinases does not occur during the HDV RNA virion assembly, and phosphorylation of the large HDAg by CKII does not confer any regulatory role in the assembly of HDV virion and empty viral particles. Our study also showed that the large HDAg without the small HDAg could efficiently assemble both monomeric and dimeric HDV genomic RNAs into secreted HBV-enveloped virus-like particles. Increasing the transfected small HDAg-expressing plasmid led to an enhancement of the packaging efficiency for the monomeric HDV genomic RNA with little effect on the packaging of dimeric HDV RNA. Similarly, HDAgs could package the trimeric HDV genomic RNA, albeit less efficiently. CsCl density gradient centrifugation confirmed that HDAgs and the monomeric and multimeric (dimer and trimer) HDV genomic RNAs formed an HBV-enveloped virus-like particle at a density of 1.23–1.25 g/ml. Thus, the assembly of the HDV virion seems to not impose much restriction on the size of HDV RNA for packaging.

Altered composition and increased endothelial cell proliferative activity of proteoglycans isolated from breast carcinoma

Background and Objectives: Proteoglycans of the extracellular matrix are vital to the growth and evolution of malignant neoplasms. The present study determined the composition of proteoglycans isolated from paired specimens of normal breast and adenocarcinoma of the breast harvested from each patient (n = 8). The proteoglycans were then tested for their ability to stimulate endothelial cell proliferation. Methods Proteoglycans were isolated by extraction with 4M guanidine hydrochloride and purified by CsCl density-gradient centrifugation. The proteoglycans were characterized and tested for their ability to simulate endothelial cell proliferation. Results In each case, the total proteoglycan content of the tumor was significantly greater than that of the corresponding normal tissue. The proteoglycans isolated from the carcinoma contained 32.2% (13.7/42.5) more chondroitin sulfate, 18.5% (5.6/30.2) less dermatan sulfate, and 29.6% (8.1/27.3) less heparan sulfate than did the proteoglycans of normal breast tissue. Proteoglycans from normal tissue did not stimulate endothelial cell proliferation, whereas those from malignant tissue stimulated proliferation by 1.3- to 1.5-fold. Conclusions These results indicate that malignant breast tissue exhibits both qualitative and quantitative changes in proteoglycan composition, which, in turn, may stimulate endothelial cell proliferation. J. Surg. Oncol. 68:250–254, 1998. © 1998 Wiley-Liss, Inc.

Altered composition and increased endothelial cell proliferative activity of proteoglycans isolated from breast carcinoma.

Proteoglycans of the extracellular matrix are vital to the growth and evolution of malignant neoplasms. The present study determined the composition of proteoglycans isolated from paired specimens of normal breast and adenocarcinoma of the breast harvested from each patient (n = 8). The proteoglycans were then tested for their ability to stimulate endothelial cell proliferation. Proteoglycans were isolated by extraction with 4 M guanidine hydrochloride and purified by CsCl density-gradient centrifugation. The proteoglycans were characterized and tested for their ability to simulate endothelial cell proliferation. In each case, the total proteoglycan content of the tumor was significantly greater than that of the corresponding normal tissue. The proteoglycans isolated from the carcinoma contained 32.2% (13.7/42.5) more chondroitin sulfate, 18.5% (5.6/30.2) less dermatan sulfate, and 29.6% (8.1/27.3) less heparan sulfate than did the proteoglycans of normal breast tissue. Proteoglycans from normal tissue did not stimulate endothelial cell proliferation, whereas those from malignant tissue stimulated proliferation by 1.3- to 1.5-fold. These results indicate that malignant breast tissue exhibits both qualitative and quantitative changes in proteoglycan composition, which, in turn, may stimulate endothelial cell proliferation.

Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili.

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Purification of Fibrillin-Containing Microfibrils and Collagen VI Microfibrils by Density Gradient Centrifugation

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. HighMrmicrofibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS–PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

The influence of intestinal mucus components on the diffusion of drugs.

Mucus, a potential diffusional barrier to drug absorption, is a complex mixture of mucin and other components. The objective of this study was to investigate the composition of native pig intestinal mucus (PIM) and the influence of identified mucus components on drug diffusion. The mucus components were separated by CsCl-density gradient centrifugation and further analyzed. The self-diffusion coefficients of mannitol, metoprolol, propranolol, hydrocortisone, and testosterone, ranging in lipophilicity from logK = -3.1 to logK = 3.3, were determined, using a small scale tracer technique. The diffusion of drugs in PIM, in solutions or dispersions of individual mucus components, and in an artificial mucus model (MLPD) reconstituted from the major mucus components mucin, lipids, protein, and DNA was compared. The dry weight of pig intestinal mucus contained (%, w/w); mucin (5%), lipids (37%), proteins (39%), DNA (6%), and unidentified materials. The most commonly occurring lipids were free fatty acids, cholesterol, and phospholipids while the most common protein was serum albumin. In PIM, but not in the purified pig gastric mucin (PPGM) solution, the diffusion of the lipophilic drugs metoprolol, propranolol, hydrocortisone, and testosterone was reduced compared to that of the hydrophilic drug mannitol. The diffusion of the lipophilic drugs was also significantly reduced in a dispersion of identified mucus lipids compared to that of mannitol. The diffusion in MLPD was similar to that in PIM for mannitol, propranolol, hydrocortisone, and testosterone, but somewhat lower for metoprolol. Lipids, rather than mucin glycoproteins, are a major component which contributes to reduced diffusion of drugs in native intestinal mucus. The results suggest that reconstituted artificial mucus models are interesting alternatives to native mucus models.

Adenovirus gene therapy for hypercholesterolemia, thrombosis and restenosis

Time for primary review 28 days. Human adenoviruses are non-enveloped, icosahedral viruses, approximately 130 nm in diameter. The linear, double-stranded DNA viral genome containing covalently attached terminal protein derived from a unique replication process is almost 36 Kb in length. Although forty nine serotypes of human adenovirus in six serological subgroups have been identified, only serotypes 2 and 5 of subgroup C have been extensively employed as gene transfer vectors. Recombinant helper-independent adenovirus vectors are easily constructed, analyzed and propagated using standard recombinant DNA and virological techniques (reviewed in [1, 2]). Foreign genes can be inserted into the adenovirus genome in a variety of locations to generate recombinant vectors, although substitution of early region 1 (E1) has been most widely used. Replacement of E1, since it is required for the efficient expression of the remainder of the viral genome, generates a vector which can only be propagated in a complementing cell line which supply the missing E1A functions in trans from an integrated copy of the appropriate fragment of the viral genome. Such E1 replacement vectors are typically propagated in the 293 [3]or 911 [4]cell lines. Recombinant viruses produced in this manner can be stably propagated to a titer of 108–109 plaque forming units per ml and are readily purified free of contaminating viral proteins and empty capsids using CsCl density gradient centrifugation for in vivo use [2]. Recombinant adenoviruses are highly efficient vectors for the transfer of foreign genes into cells both in vitro and in vivo. Because adenovirus vectors can infect a broad range of mammalian cell types, they have become the preferred vector for a wide variety of in vivo applications [5]and are currently approved for use in a limited number of human gene therapy trials. The high efficiency of … * Corresponding author. Tel.: +32 16 346182; Fax: +32 16 345990.

Self-assembly of the JC virus major capsid protein, VP1, expressed in insect cells.

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCI density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.

Specific properties of the extracellular chondroitin sulphate proteoglycans in the mandibular condylar growth centre in pigs

The developing condylar cartilage of the temporomandibular joint responds to changes in load by adaptive growth. Because local regulatory events taking place during growth processes are not well understood, investigation of extracellular matrix composition could provide new information about which matrix molecules are involved in the regulation of growth processes in this avascular tissue. The large chondroitin sulphate-rich proteoglycans in the mandibular condyle were compared to the proteoglycans in the weight-bearing femoral condyle of juvenile domestic pigs with respect to their buoyant density, chemical composition and immunological identity after isolation by dissociative extraction and CsCl density-gradient centrifugation. The distribution of these proteoglycans was studied in cryosections of mandibular condyle by immunohistochemistry using polyclonal antibodies produced against pig large proteoglycans. In the mandibular condyle, predominantly in the articular zone, the relative amount of proteoglycans with a low glycosaminoglycan content was greater than in femoral cartilage. The large proteoglycan immunologically related to aggrecan gave a protein core of 450 kDa after enzymatic deglycosylation and clearly possessed less keratan sulphate than in femoral aggrecan. Furthermore, the mandibular tissue contained another large proteoglycan with a protein core of 550 kDa after enzymatic deglycosylation, which was immunologically related to the fibroblast-like versican. Immunohistochemistry showed aggrecan increasing in amount inferiorly. In contrast, ‘versican’ was exclusively found in the fibrous and differentiation layers. Aggrecan is mainly responsible for shock absorption and versican and its homologues may be involved in the control of cell proliferation and differentiation. Thus the matrix components of the mandibular condyle seem to be adapted to its special functional needs including parallel articulation and growth.

MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions.

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrifugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.

Characterization of pig colonic mucins

Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsC1 density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The M(r) values of polymeric, reduced and digested mucins were 5.5 x 10(6), 2.1 x 10(6) and 0.6 x 10(6) respectively. This suggests that pig colonic mucin is comprised of 2-3 subunits, each subunit containing 3-4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml.g-1, 100 ml.g-1 and 20 ml.g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.

A Discontinuous Headful Packaging Model for Packaging Less Than Headful Length DNA Molecules by Bacteriophage T4

Bacteriophage T4 and other double-stranded DNA-containing bacteriophages package DNA by the classical headful packaging mechanism. In this mechanism, the packaging machinery cuts a DNA concatemer and packages a single unit length genome within the viral capsid. The length of the packaged DNA molecule is determined by the size of the viral capsid. Surprisingly, during large DNA cloning experiments, we observed that the in vitrophage T4 packaging system can package and transduce DNA molecules that are much smaller than the T4 headful size. We analyzed this phenomenon by using defined plasmid DNAs as substrates for in vitropackaging. The data showed that phage T4 can successfully package and transduce 4 to 29 kb plasmid DNA molecules. When two plasmid DNAs with different antibiotic markers were added to the packaging reaction mixture, transductants that are resistant to both the antibiotics were obtained, suggesting that both the plasmid DNAs are packaged within the same head. Analysis of the transducing particles by equilibrium CsCl density-gradient centrifugation showed that the particles have the same density as the wild-type phage. That the less than headful length molecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches. Finally, unit length plasmid DNA molecules of appropriate size were isolated from the in vitropackaged particles. Based on these data, we propose a discontinuous headful packaging model for packaging less than headful length molecules. In this model, the packaging machinery packages the first available less than headful length DNA molecule and generates a partially full head. The partially full head then reinitiates packaging on a second DNA molecule. This process continues until the head is filled with DNA.

Purification of a noncollagenous high molecular weight protein from bovine articular cartilage.

A noncollagenous high molecular weight protein (HMW) was isolated from bovine articular cartilage by CsCl density gradient centrifugation followed by DEAE-cellulose chromatography and gel filtration chromatography. The molecular weight was estimated to be 320 kDa and the reduced HMW had a molecular weight of 105 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid composition of HMW showed a high content of aspartic acid, but no hydroxylysine and hydroxyproline were detected, indicating that the protein was not a collagen. Affinity purified rabbit antibody against HMW reacted specifically with HMW, but there was no immunoreaction with cartilage-specific proteoglycan, type II collagen and type I collagen. HMW showed dose-dependent attachment to types I, II, III and V collagens. However, HMW did not bind to fibronectin, decorin and heparin. Furthermore, the binding of HMW to heat-denatured collagens was lower than native collagens. In these observation, HMW may be recognized the triple helix structure of collagen for the interaction.

DNA Structure Determines Protein Binding and Transcriptional Efficiency of the Proenkephalin cAMP-responsive Enhancer

Two precisely arranged proenkephalin cAMP response elements (CREs) behave as a single protein binding site. The experiments described support a model in which a secondary structural change creates a new binding site, which is made up of sequences from both of the CREs. The CRE-binding protein (CREB) binds CRE-1, but binding there is entirely dependent on the presence of CRE-2. Electron spectroscopic images show that a CREB dimer occupies twice as much DNA in the proenkephalin gene as in the prodynorphin gene. The enhancer region is sensitive to P1 nuclease in a CREB concentration-dependent manner, and sensitivity is strand-specific, indicating protein-stabilized structural change. DNase I analysis shows that in the native proenkephalin gene, CREB binds both CRE-1 and CRE-2. In vivo, both CREs are occupied in the transcriptionally active proenkephalin gene, while neither is in the silent gene. Whereas CREB can bind CRE-2, mutation or elimination of either proenkephalin CRE alters response to second messengers and transcription factors. Thus, binding to CRE-2 alone is not sufficient. Specific and efficient transcription of the proenkephalin gene requires the presence of both CREs, precisely arranged to allow them to form a single protein binding site.

Composition of human airway mucins and effects after inhalation of acid aerosol

Characterization of normal airway mucus is required to elucidate mechanisms protecting the airways and to understand changes associated with disease and environmental insult. Toward this goal, we collected bronchial washes (10 ml saline) from healthy human subjects to 1) evaluate the yield of high-density material (delta > or = 1.35 g/ml), and 2) characterize glycoconjugates associated with collected secretions. Samples were lipid extracted followed by CsCl density gradient centrifugation. The yield of high-density material from individual subjects was variable but sufficient to demonstrate that mucin glycoproteins are a major constituent of mucus from healthy airways and that proteoglycans are absent. Next, we investigated whether inhalation of H2SO4 aerosol (1,000 microgram/m3), an environmental insult associated with alterations in mucociliary clearance, changes the composition of high-density glycoproteins in airway secretions. In a paired, double-blinded study, high-density fractions of bronchial secretions from 12 subjects were collected 18 h after exposures of 2 h to aerosolized NaCl and H2SO4. In all cases the high-density material displayed characteristics of mucin glycoproteins. In addition, a unique 150-kDa glycoprotein was detected in most but not all samples and may represent a small mucin glycoprotein differentially expressed in humans. No differences were noted between the two exposure conditions in the profiles of the glycoproteins or proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Statistically, large changes with acid exposure in the composition of carbohydrates and amino acids were absent. Thus no substantial systematic changes in airway mucin glycoproteins or closely associated proteins and glycoproteins were correlated with H2SO4 exposure. Alternatively, statistical analysis of the differences between exposures in glycoprotein constituents among subjects denoted greater variability in carbohydrates compared with amino acids with repeated sampling, suggesting normal daily variations in the mucin composition of individual airway mucus.

Influence of Plasmids on Catalase and Superoxide Dismutase Activities in Listeria monocytogenes

Two of nine strains of Listeria monocytogenes examined were found to contain plasmid DNA. Strain 19112 contained a 31.1 kb plasmid and strain 7644 contained a 49.4 kb plasmid. Each of the strains was cured of its plasmid by heat treatment at 46°C. Both the wild-type and plasmid-negative forms of each strain were screened for cadmium resistance. The 31.1 kb plasmid of L. monocytogenes 19112 was shown to confer resistance to cadmium. Listeria monocytogenes 7644 did not show resistance to cadmium. The plasmids for both strains were isolated and purified by CsCl density-gradient centrifugation. Each plasmid was electroporated back into the respective plasmid-negative strain. The catalase (CA) and superoxide dismutase (SOD) activities were determined for the wild type, plasmid-negative and electroporated strains. There was a significant decrease in CA and SOD activities upon loss of the plasmid from each strain of L. monocytogenes. Strain 19112 showed a 36% decrease in CA activity and an 81% decrease in SOD activity as a result of plasmid removal. Strain 7644 showed a 22% decrease in both CA and SOD activities following plasmid loss. Catalase and SOD activity levels increased for both strains following reinsertion of the plasmid through electroporation. Catalase and SOD activity levels of L. monocytogenes 7644 were higher for the transformed strain than those of the wild type. Catalase and SOD activity levels of transformed L. monocytogenes 19112 were less than in the corresponding wild type. It appears that plasmids in L. monocytogenes strains 19112 and 7644 may be involved in influencing the regulation of the production of CA and SOD. Plasmid copy number may influence the level of activity of these enzymes.

Purification, characterization and developmental expression of a brain-specific chondroitin sulfate proteoglycan, 6B4 proteoglycan/phosphacan.

A large brain-specific chondroitin sulfate proteoglycan, identified with monoclonal antibody 6B4 (6B4 proteoglycan/phosphacan), was isolated from rat brain. Soluble proteoglycans in the phosphate-buffered saline extract from 20-day-old rat whole brain were fractionated by anion exchange chromatography and CsCl density gradient centrifugation. 6B4 proteoglycan was further purified by gel filtration and additional ion exchange chromatography. The molecular mass of 6B4 proteoglycan shifted from 800 to 300 x 10(3) mol. wt after chondroitinase ABC digestion. The core protein was substituted with chondroitin sulfate chains with an average molecular weight of 21,000, keratan sulfate and HNK-1 carbohydrates. Glycosidase digestion of 6B4 proteoglycan with O-glycanase, N-glycanase, endo-beta-galactosidase, or keratanase did not remove the HNK-1 epitopes. The expression of 6B4 proteoglycan was developmentally regulated in the rat cerebral cortex; appearing first at embryonic day 14, peaking at postnatal day 0, and persisting throughout adulthood at a lower level. Immunohistochemical analysis indicated that 6B4 proteoglycan was distributed along the radial glial fibers and on the migrating neurons in the embryonal rar cerebrum. The radial glial fibers were stained intensely all along their length, but the neurons in the cortical plate were not stained in contrast to the moderate staining of the migrating neurons in the intermediate zone and the subplate. From postnatal day 5 to postnatal day 20, 6B4 proteoglycan was present throughout the cortex. After postnatal day 30, staining of the neuropil was weakened, and the expression of 6B4 proteoglycan was restricted around subsets of neurons. The positive neurons were mostly non-pyramidal cells (> 95%) and were relatively concentrated in layers IV and VI of the primary somatosensory cortex. Immunohistochemical analysis of the dissociated cortical neurons indicated that 6B4 proteoglycan was distributed on the cell bodies and neurites. 6B4 proteoglycan strikingly promoted neurite extension of cortical neurons from embryonic day-16 rat embryos when coated on coverslips as a substrate. 6B4 proteoglycan is a brain-specific chondroitin sulfate proteoglycan which carries keratan sulfate and HNK-1 carbohydrates. The spatiotemporal expression profile and effects on the dissociated cerebral neurons suggest that 6B4 proteoglycan plays important roles in the migration and differentiation of neurons in the immature cortex, and also in the maintenance of subsets of neurons in the mature cortex.

Purification and Biochemical Characteristics of the Rabbit Haemorrhagic Disease Virus

The causative rabbit haemorrhagic disease viral antigen (RHDV) was purified by sucrose and caesium chloride density gradients. The viral fractions were characterised by a haemagglutination test and a dodecylsulfat polyacrilamide gel electrophoresis (SDS-PAGE). Different migration patterns were observed after sucrose and caesium chloride (CsCl) density gradient centrifugations.

Multiple receptor-like protein tyrosine phosphatases in the form of chondroitin sulfate proteoglycan

The possibility that some of the brain proteoglycans are receptor-like protein tyrosine phosphatases (PTPases) was investigated. Membrane-bound proteoglycan fractions were prepared from the postnuclear membrane fraction of 8-day-old rat brain by DEAE ion-exchange chromatography and CsCl density gradient centrifugation. The isolated proteoglycan fractions showed high PTPase specific activities together with the typical PTPase characteristics. Renaturation experiments indicated that chondroitin sulfate proteoglycans with 380- and 170-kDa core proteins carried the PTPase activity. The proteoglycan with 380-kDa core protein was identified as RPTPβ/ζ bearing HNK-1 carbohydrate.

Preparation of Genomic DNA from Plant Tissue

This unit describes two methods for preparing genomic DNA from plant tissue. In the first method, plant cells are lysed with ionic detergent, treated with protease, and subsequently purified by cesium chloride (CsCl) density gradient centrifugation. The second method is based upon a series of treatments with the nonionic detergent cetyltrimethylammonium bromide (CTAB) to lyse cells and purify nucleic acid. Nucleic acid is recovered from the final CTAB solution by isopropanol or ethanol precipitation. The first method, although somewhat more lengthy, results in highly purified nucleic acid. The second method requires fewer manipulations, results in very high yields (approximately 10-fold higher per gram fresh tissue depending on species and condition of starting material), and produces DNA that is less pure but nonetheless suitable in quality for use in many molecular biology manipulations.