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141.
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
142.
143.
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
144.
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
145.
Theresa A. Grebe William K. Seltzer Jean DeMarchi Dinithi K. Silva W. W. Doane David Gozal S. F. Richter C. Michael Bowman R. A. Norman Susan N. Rhodes Lucy S. Hernried Shirley Murphy Ivan R. Harwood Frank J. Accurso Karen D. Jain 《American journal of human genetics》1994,54(3):443-446
We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling. 相似文献
146.
Michael Dean J. Claiborne Stephens Cheryl Winkler Deborah A. Lomb Mark Ramsburg Raleigh Boaze Claudia Stewart Lauren Charbonneau David Goldman Bernard J. Albaugh James J. Goedert R. Palmer Beasley Lu-Yu Hwang Susan Buchbinder Michael Weedon Patricia A. Johnson Mary Eichelberger Stephen J. O'Brien 《American journal of human genetics》1994,55(4):788-808
A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies. 相似文献
147.
Richard W. Hyman Susan Frank Craig H. Warden Aaron Daluiski Renu Heller Aldons J. Lusis 《Biochemical genetics》1994,32(11-12):397-407
Quantitative trait locus (QTL) analysis is a statistical method that can be applied to identify loci making a significant impact on a phenotype. For the phenotype of susceptibility to diet-induced atherosclerosis in the mouse, we have studied four quantitative traits: area of aortic fatty streaks and serum concentrations of high-density lipoprotein-bound cholesterol (HDL-cholesterol), apolipoprotein A-I, and apolipoprotein A-II (apo A-II). QTL analysis revealed a significant locus on chromosome 1 distal impacting serum apo A-II concentration on a high-fat diet and serum HDL-cholesterol concentration on a chow diet. This locus is presumablyApoa-2, the structural gene for apo A-II. QTL analysis of aortic fatty streaks failed to reveal a significant locus. 相似文献
148.
Morphological variability of geographically distinct populations of the estuarine copepod Acartia Tonsa 总被引:1,自引:1,他引:0
Garmew Tanya G. Hammond Susan Mercantini Amy Morgan Jessica Neunert Cindy Fornshell John A. 《Hydrobiologia》1994,(1):149-156
Variations in the number of spines on the left and right posterior dorsal and posterior margins of the prosome as well as the length of the prosome of Acartia tonsa from three estuaries, the upper western side of the Chesapeake Bay, Montauk Bay near the eastern end of Long Island Sound and the coast of Peru were determined. The length of the prosome and number of spines in each of the four locations were used as an indication of morphological similarity between the populations. 相似文献
149.
150.
Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes 总被引:1,自引:0,他引:1
Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by Gl/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the Gl/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABAB receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low Km GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [3H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other Gl/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions. 相似文献