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161.
P. MOLLOY, L. BRYDON, A.J, PORTER AND W.J. HARRIS. 1995. New methods to quantitatively remove bacteria from food and water samples are required to meet modern safety standards. The recent development of techniques to make Fab/Fv/scFv fragments in bacteria has provided the opportunity to exploit antibodies as specialized chemicals for affinity removal technologies. Single-chain fragments against Pseudomonas aeruginosa have been expressed in Escherichia coli , purified via a fused poly-histidine tail and immobilized upon polystyrene beads. The resulting immunoaffinity columns have been shown to effectively remove greater than 90% of an applied 10 million bacteria after a single passage through the column. Column material in the absence of single-chain retained less than 10% of the bacteria. Pseudomonas were also removed from milk, mixed bacterial cultures and when present at low cell densities. 相似文献
162.
Susan H. McKiernan Murray K. Clayton Barry D. Bavister 《Molecular reproduction and development》1995,42(2):188-199
Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc. 相似文献
163.
164.
Sheri L. Holmen Matt W. Vanbrocklin Robert R. Eversole Susan R. Stapleton Leonard C. Ginsberg 《In vitro cellular & developmental biology. Animal》1995,31(5):347-351
Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids,
however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available
cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study
include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium
methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic
(EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these
vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio.
Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current
theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection
studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes
comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions
for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration
of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical,
easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes. 相似文献
165.
166.
Peter E. Molloy Barbara M. Graham Pauline M. Cupit Steven D. Grant Andrew J. R. Porter Charles Cunningham 《Molecular biotechnology》1995,4(3):239-245
This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity
or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain
their proper conformation. 相似文献
167.
Gill JH Molloy CA Shoesmith KJ Bayly AC Roberts RA 《Cell death and differentiation》1995,2(3):211-217
The responses of a series of rat hepatoma cell lines (FaO, HTC, RH1) to the rodent non-genotoxic hepatocarcinogen and per-oxisome proliferator (PP) Nafenopin were studied to determine if this PP acts with EGF, a naturally occurring liver growth regulator, to perturb the balance between mitosis and apoptosis. EGF enhanced the growth of FaO cells (well differentiated) and HTC cells (intermediate differentiation) but not of the poorly differentiated RH1 cell line. Nafenopin also increased FaO cell growth but, surprisingly, retarded the growth of both HTC and RH1 cells. Since population expansion kinetics result from mitosis and death, replicative DNA synthesis (RDS) and apoptotic cell death were measured in HTC cells. As expected, EGF elevated RDS and suppressed cell death. However, Nafenopin depressed HTC net population expansion via a suppression of cell death coupled to a more marked inhibition of RDS. This apparent paradox was investigated using soft agar cloning. This revealed sub-populations with differing growth kinetics suggesting selective clonal expansion via an alteration in the balance between mitosis and apoptotic cell death. At later stages, cells are refractory to EGF and Nafenopin, suggesting that genetic changes may have superseded such factor-dependence. 相似文献
168.
Stephanie Wengert Watts Susan L. Gackenheimer Donald R. Gehlert Marlene L. Cohen 《Neurochemistry international》1994,24(6):565-574
Although the density and distribution of 5-HT2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized 125I-lysergic acid diethylamide ([125I]LSD) to quantify and compare 5-HT2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [125I]LSD binding from 5-HT2A and D2 binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist. 相似文献
169.
The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T3 (0.6 g/Kg) once a day and T4 (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma. 相似文献
170.
Gap junction-mediated intercellular communication was analyzed in a model system in which tissue necrosis and remodeling could be modulated. This in vitro system, previously used for analysis of epithelial-mesenchymal tissue interaction, was modified to permit analysis of the presence and extent of intercellular communition by monitoring intercellular transfer of the micro-injected fluorescent dye, Lucifer Yellow. Light and transmission electronmicroscopy were employed to correlate the presence and degree of gap junctional communication (coupling) with tissue morphology. Digital image analysis was used to determine cell density and mitotic indices within the outgrowths of explants. Our results indicated that cell communication in outgrowths adjacent to necrotic foci within an explant was minimal or absent. Cell-coupling in outgrowths adjacent to a compartment of viable mesenchyme was significantly higher-equivalent to unseparated control cultures. A time-course study demonstrated correlation of increased levels of cell-coupling in outgrowths with the level of tissue remodeling within an explant. Our conclusions from these studies are that embryonic mesenchymal cell populations may be selectively uncoupled as a result of alterations in the microenvironment produced by a proximate impaired cell population. It is proposed that endogenous factors in the microenvironment (wound signals), emanating from impaired cell populations, regulate gap junction-mediated intercellular communication in adjacent viable tissue. Normal, unimpaired populations of cells surrounding an area of injury are thereby isolated from the effects of a potentially toxic environment. This could serve as a protective function in development and may represent, in a more general sense, part of the repertoire of events associated with tissue repair and remodeling. 相似文献