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Showing papers on "Lysis published in 1994"


Journal ArticleDOI
TL;DR: Improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate-treated sediment rich in organic matter are reported.
Abstract: This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS) Images

417 citations


Journal ArticleDOI
TL;DR: The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity and was able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions.
Abstract: This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD. The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

372 citations


Journal ArticleDOI
TL;DR: This unit describes two methods for preparing genomic DNA from plant tissue that requires fewer manipulations, results in very high yields, and produces DNA that is less pure but nonetheless suitable in quality for use in many molecular biology manipulations.
Abstract: 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.

223 citations


Journal ArticleDOI
TL;DR: There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens and that destruction of gram- negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.
Abstract: High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane. Images

170 citations


Journal ArticleDOI
01 Oct 1994-Yeast
TL;DR: An easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate–gel electrophoresis and Western blotting, applicable to the study of large numbers of samples in 1 day.
Abstract: We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)-gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.

166 citations


Journal ArticleDOI
TL;DR: KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan, and both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins.
Abstract: A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.

139 citations


Journal ArticleDOI
TL;DR: The results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.
Abstract: The mechanism of the initial steps of bacteriophage infection in Lactococcus lactis subsp. lactis C2 was investigated by using phages c2, ml3, kh, l, h, 5, and 13. All seven phages adsorbed to the same sites on the host cell wall that are composed, in part, of rhamnose. This was suggested by rhamnose inhibition of phage adsorption to cells, competition between phage c2 and the other phages for adsorption to cells, and rhamnose inhibition of lysis of phage-inoculated cultures. The adsorption to the cell wall was found to be reversible upon dilution of the cell wall-adsorbed phage. In a reaction step that apparently follows adsorption to the cell wall, all seven phages adsorbed to a host membrane protein named PIP. This was indicated by the inability of all seven phages to infect a strain selected for resistance to phage c2 and known to have a defective PIP protein. All seven phages were inactivated in vitro by membranes from wild-type cells but not by membranes from the PIP-defective, phage c2-resistant strain. The mechanism of membrane inactivation was an irreversible adsorption of the phage to PIP, as indicated by adsorption of [35S] methionine-labeled phage c2 to purified membranes from phage-sensitive cells but not to membranes from the resistant strain, elimination of adsorption by pretreatment of the membranes with proteinase K, and lack of dissociation of 35S from the membranes upon dilution. Following membrane adsorption, ejection of phage DNA occurred rapidly at 30°C but not at 4°C. These results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.

109 citations


01 Oct 1994
TL;DR: The results suggest that dimeric forms of antibodies could be useful for converting cells with low density antigens into useful targets for therapy.
Abstract: Antibody-mediated lysis of cells involves a complex interaction between the cell, the target antigen, the antibody and host effector mechanisms. One such mechanism, complement-mediated cell lysis, requires the interaction of C1q with the antibody heavy chain constant regions, and in particular the CH2 domain. Here we investigate the potential benefit of multiple-domain forms of the therapeutic monoclonal antibody CAMPATH-1H. This antibody is directed against the CDw52 antigen expressed by human lymphocytes and has proven lytic abilities both in vitro and in vivo. Using target cells with either high or low antigen density, engineered antibodies that contained additional domains in tandem (CH2, hinge-CH2 or Fc intramolecular repeats) showed no improvement in complement-mediated lysis when compared with controls. However, a homodimeric form of the antibody that was engineered by mutation of a serine residue to cysteine near the carboxy-terminal of the CH3 domain, exhibited markedly improved lysis using target cells expressing antigen at low density. Interestingly, no improvement was seen using cells expressing antigen at high density. These results suggest that dimeric forms of antibodies could be useful for converting cells with low density antigens into useful targets for therapy.

98 citations


Journal ArticleDOI
TL;DR: The measurement of LDH activity in vitro provides a sensitive, accurate and cost-effective alternative to the use of either radioisotopic or dye-based assays for the determination of cell numbers.

94 citations


Journal Article
TL;DR: CTL-mediated lysis of cells infected with an intracellular bacterium that inhibits lysosomal fusion and is confined to an endosomal vacuole is demonstrated, which implicate endogenous Ag processing for Chlamydia-specific cytolysis.
Abstract: Intracellular bacterial pathogens have evolved to either grow in the nutrient-rich cytoplasm or remain sequestered within a vacuole. One potentially important selective advantage for growth within a vacuole may be evasion of cell-mediated detection and cytolysis. To address this question we used the endosomally confined bacterium Chlamydia trachomatis, which naturally infects epithelial cells, to examine CTL-mediated lysis of nonphagocytic cells. CTL-mediated lysis of infected target cells was detected, although the increased expression of ICAM-1 by transfection was required. The elimination of CD8+ T cells or addition of brefeldin A or cycloheximide eliminated specific cytolysis, whereas conversely, treatment with chloroquine or ammonium chloride had only minor effects. These results implicate endogenous Ag processing for Chlamydia-specific cytolysis. This work demonstrates CTL-mediated lysis of cells infected with an intracellular bacterium that inhibits lysosomal fusion and is confined to an endosomal vacuole.

90 citations


Journal ArticleDOI
TL;DR: It is concluded that a portion of GRA3 secreted from the parasite as a soluble protein directly inserts into the vacuole membrane in an oligomeric form, the first report in Toxoplasma or related parasites of a protein which inserts into that membrane for some purpose other than to lyse that membrane.

Journal ArticleDOI
TL;DR: The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.

Journal Article
TL;DR: The murine CTL hybridoma PMMI was used, activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CTLs in the absence of perforin, and it was found that PMMI is equipped with membrane TNF-alpha as a potential lytic mechanism, but T NF-alpha is unlikely to be involved in acute lytic reactions.
Abstract: The murine CTL hybridoma PMMI has been shown by the most sensitive techniques to be devoid of perforin. We thus used PMMI activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CTLs in the absence of perforin. We found that PMMI is equipped with membrane TNF-alpha as a potential lytic mechanism, but TNF-alpha is unlikely to be involved in acute (4 h) lytic reactions. On the other hand, PMMI readily lyses target cells expressing the gene for the Fas Ag, but does not lyse target cells expressing fas antisense DNA. The generation of fas-dependent lysis required protein synthesis in PMMI, but target cell protein synthesis was not required for lysis. Lysis of Fas-positive target cells by PMMI was accompanied by DNA fragmentation, and both lysis and DNA fragmentation were blocked by inhibition of protein synthesis in the effector cell. We find the relative extent and kinetics of fas-dependent lysis and DNA fragmentation indistinguishable from that seen in "classical" CTL lytic assays. Both fas- and perforin-dependent lysis were blocked by inhibitors of poly(ADP) ribosylation. We found very little difference in the sequence of events in target cells lysed by the fas pathway compared with the classical (probably perforin) lytic pathway. Given the widespread distribution of fas, particularly in hematopoietic target cells, caution may be required in interpreting the relationship between parameters such as DNA fragmentation and 51Cr-release solely on the basis of the granule exocytosis model.

Journal ArticleDOI
TL;DR: The results suggest that not only LPS but also surface proteins probably play important roles in T. ferrooxidans adhesion to solid surface, as determined by an increasedadhesion to hydrophobic sulfur prills and C-dodecanoic acid binding.
Abstract: Conditions for the partial removal of lipopolysaccharide (LPS) from Thiobacillus ferrooxidans are described. Raising the pH of the solution containing the cells from pH 1.5 to pH 6.8 to 8.0 releases about 50% of the LPS without cell lysis. The release of LPS begins at pH 3.5, and it was not affected by EDTA concentration. Partial removal of LPS exposed higher amounts of a 40-kDa outer membrane protein in the bacteria, as detected by a dot immunoassay employing an antiserum against the T. ferrooxidans surface protein. This higher protein exposure and the reduced LPS content increased the hydrophobicity of the cell surface, as determined by an increased adhesion (50%) to hydrophobic sulfur prills and C-dodecanoic acid binding (2.5-fold) compared with control cells. In addition, adhesion of radioactively labeled microorganisms to a sulfide mineral was inhibited (40%) in the presence of previously added LPS. Our results suggest that not only LPS but also surface proteins probably play important roles in T. ferrooxidans adhesion to solid surfaces.

Journal ArticleDOI
TL;DR: The lysis system involving pDKL02 is useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point.
Abstract: Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.

Journal ArticleDOI
TL;DR: The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.
Abstract: The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

Patent
08 Jul 1994
TL;DR: In this article, a method for purifying from cells a target compound such as nucleic acid is provided, which comprises the following steps: 1) lysing a cell suspension to form a cell lysate containing the target compound, 2) applying the cell lysis to a filter to remove unwanted cells and cell debris, 3) contacting the filtered lysatter with a solid phase matrix under conditions to bind the nucleic acids to the matrix, 4) separating the resultant filtered lysisate from the matrix; and 5) eluting the target compounds from the matrices
Abstract: A method is provided for purifying from cells a target compound such as nucleic acid. The method comprises the following steps: 1) lysing a cell suspension to form a cell lysate containing the target compound; 2) applying the cell lysate to a filter to remove unwanted cells and cell debris; 3) contacting the filtered lysate with a solid phase matrix under conditions to bind the nucleic acid to the matrix; 4) separating the resultant filtered lysate from the matrix; and 5) eluting the target compound from the matrix. Apparatus is also provided. Complex purification procedures such as centrifugation are avoided.


Journal ArticleDOI
TL;DR: Exposure of isolated bovine neutrophils to partially purified Pasteurella haemolytica leukotoxin caused increased synthesis ofleukotriene B4 (LTB4) but not thromboxane B2 (TXB2) from endogenous arachidonic acid.
Abstract: Exposure of isolated bovine neutrophils to partially purified Pasteurella haemolytica leukotoxin caused increased synthesis of leukotriene B4 (LTB4) but not thromboxane B2 (TXB2) from endogenous arachidonic acid. Synthesis of LTB4 was closely correlated with leukotoxin-induced neutrophil lysis. At low toxin concentrations, LTB4 production lagged behind leukotoxin-induced neutrophil lysis over a 3-h period. The neutralizing monoclonal antileukotoxin antibody MM601 neutralized both leukotoxin-induced neutrophil lysis and LTB4 synthesis. Both leukotoxin-induced neutrophil lysis and LTB4 synthesis were Ca(2+)-dependent. When leukotoxin-induced LTB4 synthesis from exogenous arachidonic acid was examined, significant LTB4 synthesis occurred at 5 min of leukotoxin exposure, which was before leukotoxin-induced lysis developed. Leukotoxin-induced LTB4 synthesis from endogenous arachidonic acid appears to require leukotoxin-induced plasma membrane damage (occurring during neutrophil lysis), whereas LTB4 synthesis from exogenous arachidonic acid is initiated rapidly and occurs in the absence of plasma membrane damage.

Patent
12 Apr 1994
TL;DR: In this article, a chimeric compound that contains a cell-specific ligand linked to a pore-forming agent capable of lysing a cell is described, and the pore forming agent is shown to be effective in cell lysis.
Abstract: A chimeric compound that contains a cell-specific ligand linked to a pore-forming agent capable of lysing a cell.

Journal ArticleDOI
TL;DR: The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half‐life of 57 h at 37°C, and potentially leading to extracellular modification of glycoprotein oligosaccharides.
Abstract: We have previously demonstrated that Chinese hamster ovary (CHO) cell lysates harbor sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities that can accumulate extracellularly in CHO cell culture, thereby potentially leading to extracellular modification of glycoprotein oligosaccharides. The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half-life of 57 h at 37 degrees C.We have extended this work to determine whether 293, NS0, or hybridoma cell lysates contain similar glycosidase activities. The pH-activity profiles of beta-galactosidase and beta-hexosaminidase in lysates of these three cell lines resemble the pH-activity profiles for these enzymes in CHO cell lysate, whereas the pH-activity profiles of sialidase and fucosidase appear to be cell-type dependent. Sialidase activities were relatively stable at pH 4.5 in 293, NS0, and hybridoma cell lysates. However, the activities in 293 and NS0 cell lysates were unstable at pH 7.5, with no activity remaining after a 2-h incubation at 37 degrees C. The sialidase activity in hybridoma cell lysate was moderately stable at pH 7.5 with 30% of the activity remaining after a 2-h incubation at 37 degrees C. We conclude that the sialidase activites from 293, NS0, and hybridoma cells have characteristics similar to the vast majority of reported mammalian sialidase activities, and that these activities are markedly differant from the CHO cell sialidase activity.Finally, sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities were measured at pH 7 in cell-free bioreactor supernatants of the hybridoma cell line. As previously observed in CHO cell culture, all four glycosidase activities were present in the hybridoma supernatants. However, the sialidase activity in hybridoma supernatant was an order of magnitude lower than in CHO cell culture supernatant despite the fact that the hybridoma cell lysis rate was an order of magnitude higher. (c) 1994 John Wiley & Sons, Inc.

Patent
26 Aug 1994
TL;DR: A stool lysis buffer having a high concentration of buffer, a chelating agent, and a salt has the ability to lyse mammalian cells but not bacterial cells as discussed by the authors.
Abstract: A stool lysis buffer having a high concentration of buffer, a chelating agent, and a salt has the ability to lyse mammalian cells but not bacterial cells. Lysed cells release nucleic acids into the buffer. A particulate fraction is removed which includes the unlysed bacterial cells.

Journal ArticleDOI
TL;DR: The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis.
Abstract: Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis.

Journal ArticleDOI
TL;DR: New procedures were incorporated into established direct lysis methods in order to improve the quality of DNA recovered from sediments, and DNA purified using ion exchange chromatography was comparable in quality to that recovered from CsCl gradient ultracentrifugation, and was obtained at substantial savings in time and expense compared to the latter method.

Journal Article
TL;DR: It is found that the complement proteins C4, C3d, C5b-9, and properdin bind to rgp 120-coated CD4+ T cells of healthy individuals when incubated in autologous serum.
Abstract: The mechanism of CD4+ cell depletion in HIV-infected patients is poorly understood. In this study we investigated whether rgp120 can activate the complement system in the absence of anti-gp120 Abs. We found that the complement proteins C4, C3d, C5b-9, and properdin bind to rgp 120-coated CD4+ T cells of healthy individuals when incubated in autologous serum. Activation of the complement system occurred primarily via the classical pathway and was abolished in sera deficient in C1q and C4 as well as in the presence of EDTA. No cell lysis was observed in a lymphocytotoxicity assay using human serum, possibly because of homologous restriction of complement lysis. In contrast, addition of rabbit sera induced lysis of the rgp 120-precoated cells. Cell lysis by rabbit serum was found to be because of naturally occurring IgM anti-gp 120 Abs. The rgp 120, which was immobilized on the surface of microtiter plates activated complement in the absence of lymphocytes. Complement activation by cell-bound HIV-1 envelope glycoprotein gp120 with subsequent opsonization may be relevant for the elimination of noninfected CD4+ T cells in HIV-infected patients.

Journal ArticleDOI
TL;DR: The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule, and is located in the middle of a putative membrane‐spanning helical domain of S, near the sites of other S− mutations with recessive non‐lytic phenotypes.
Abstract: The S and R genes of the bacteriophage lambda are required for lysis of the host. R encodes 'endolysin', a soluble transglycosylase which accumulates in the cytoplasm during late protein synthesis. S encodes a 'holin', a small membrane protein which, at a precisely scheduled time, terminates the vegetative cycle by forming a lethal lesion in the membrane through which gpR gains access to the peptidoglycan. A missense allele of S, Ala52Gly, causes lysis to occur prematurely at about 19-20 min after induction of a lysogen, compared to 45 min for the wild type. This allele has a severe plaque-forming defect which appears to be entirely a consequence of the early lysis and resultant severe reduction in particle burst size. The early-lysis phenotype is dominant and is aggravated, in terms of an even more reduced burst size, at both 30 degrees C and 42 degrees C. The mutation maps in the middle of a putative membrane-spanning helical domain of S, near the sites of other S- mutations with recessive non-lytic phenotypes. The mutation has no effect on S-protein accumulation or on the ratio of S107 and S105 products in the membrane. The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule.

Journal ArticleDOI
TL;DR: It is concluded that AD202 is one of the most appropriate strains for the purpose of producing GST-fusion proteins in E. coli and results suggest that an extremely unstable fusion protein such as GSTCDS f can be stabilized in AD202.
Abstract: Recombinant proteins prepared by genetic engineering are utilized for various purposes in molecular biology such as production of specific antibodies or investigation of the mechanism of protein—DNA or protein-protein interactions (1). There are several methods commonly used to produce recombinant proteins in bacteria or insect cells (2). Expression of a recombinant protein in E. coli as a fusion protein with glutathione S-transferase (GST) is one of the most popular and easiest methods (3). However, we often encounter the serious problems that some fusion proteins are rapidly degraded or cannot be solubilized in bacterial cells. To prepare a polyclonal antibody against the 7 chain of the high affinity IgE receptor (FCCRIY), we constructed GST-FceRI-y, which was composed of the GST gene joined to the cytoplasmic portion of the FceRty cDNA. In initial experiments, we utilized two commonly used bacteria strains, DH5a and TGI, for production of the fusion protein. Although the GST-fusion protein was induced with isopropyl-/3-D-thiogalactopyranoside and solubilized in the supernatant of the bacterial lysate after sonication, the fusion protein was rapidly cleaved at the fusion joint between the GST and FwRI-y during purification (Fig l.A). This cleavage could not be prevented by the addition of several available protease inhibitors such as phenylmethyl sulfonyl fluoride, aprotinin, leupeptin, bestatin and ethylenediaminetetraacetic acid (data not shown). Several trials for alternative methods such as shortening the induction time, changing the incubation temperature, or lysis with freezing and thawing method instead of sonication did not improve the results at all (data not shown). To overcome this problem, we then used several protease negative E.coli strains (Table 1). In one of these strains, AD202, which is defective in ompT encoding an outer membrane-associated protease (4), the fusion protein was not cleaved at all and recovered from bacterial lysate with an expected molecular size (Fig. 1A). The prevention of degradation was observed not only with this construct but also with other GST-fusion proteins including GST-CD3e (Fig. IB). It is noteworthy that a fusion protein, GST-CD3f, which could not be detected in DH5a, could be induced and purified by using AD202 (Fig. 1C). These results suggest that an extremely unstable fusion protein such as GSTCDS f can be stabilized in AD202. We next examined the efficiency of this strain by using a larger fusion protein construct. A 75 kD GST-CTBPl, which contains a coding frame of a singlestranded DNA and RNA binding protein of the yeast, was purified efficiently in AD202, whereas most of the products were degraded in DH5a (Fig. ID). This suggests that AD202 is also effective for fusion proteins as large as 75 kD and for those encoding nucleic acid binding proteins. From these observations, we conclude that AD202 is one of the most appropriate strains for the purpose of producing GST-fusion proteins in E. coli.

Journal ArticleDOI
TL;DR: In one day, this method yields viral antigen with minimal cellular contaminants, in a concentrated volume suitable for subsequent biochemical, vaccine or diagnostic uses.

Journal ArticleDOI
TL;DR: To study the feasibility of designing polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intrACEllular drug delivery, a covalent conjugate of a poly(amidoamine) (MBI) was prepared.
Abstract: Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml).