Target substrate-specific hammerhead ribozyme cleaves the specific mRNA and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To modulate apoB gene expression, we designed hammerhead ribozymes targeted at AUA 6665 2 and GUA 6679 2 of apoB mRNA, designated RB16 and RB15, respectively, and investigated their effects on apoB mRNA in HepG2 cells. The results demonstrated that RB15 and RB16 ribozyme RNAs cleaved apoB RNA efficiently in vitro. Both ribozymes, RB15 and RB16, were used to construct recombinant adenoviral vectors, designated AvRB15 and AvRB16, respectively, for in vivo gene transfer. HepG2 cells were infected with 2 ؋ 10 5 plaque-forming units of AvRB15 for 5, 10, 15, and 24 h. An RNase protection assay showed that the expression of the RB15 transcript was time-dependent; it increased ϳ300-fold from 5 to 24 h. Using reverse ligation-mediated polymerase chain reaction, the 3 cleavage product of apoB mRNA was detected, and the exact cleavage site of apoB mRNA was confirmed by sequencing. Importantly, the levels of apoB mRNA in HepG2 cells decreased ϳ80% after AvRB15 infection. Pulse/chase experiments on HepG2 cells treated with AvRB15 and AvRB16 demonstrated that ribozyme cleavage produced a truncated protein that was secreted at a density of 1.063-1.210 g/ml. The cleavage activity of RB15 on apoB mRNA was more efficient than that of RB16. Moreover, pulse/chase experiments in HepG2 cells treated with AvRB15 revealed that most of the truncated apoB protein was degraded intracellularly. We conclude that hammerhead ribozyme targeted at GUA 6679 2 of apoB mRNA cleaves apoB mRNA, results in decreased apoB mRNA levels, and generates a truncated apoB of the expected size in vivo. Thus, the therapeutic application of ribozyme in regulating apoB production holds promise.
Hepatic lipase is an enzyme which hydrolyzes triglycerides from plasma lipoproteins and thus takes part in the metabolism of intermediate density lipoproteins and high-density lipoproteins. The search described here concentrated on mutations of the HL gene in 129 patients with combined hypertriglyceridemia/hyperalphalipoproteinemia and in 184 members of 19 families with familial combined hyperlipidemia. Controls were 100 subjects with favorable lipid values (age 46-51 years). Mutation screening and analysis were performed by temperature-gradient gel electrophoresis, allele-specific restriction genotyping, and sequencing. Six different missense mutations and four different silent mutations were found in the HL gene. The alleles Phe-267 and Gln-343 were detected only once in the patient group with hypertriglyceridemia and hyperalphalipoproteinemia and were not detected in the control group. The allele Met-383 was rare in both patients and controls. We found 9.3% of the patients and only 3.0% of controls to be carrying the Val-73-Met missense mutation. The allele Phe-334 was found in 5.43% of patients and in 2.0% of controls. The difference between the frequencies of these alleles was significant between male patients and male controls (Met-73 P=0.044; Phe-334 P=0.047). Also, the summarized odds ratio of 3.28 (95% confidence interval 1.23-8.73) demonstrates that mutation carriers are significantly more prevalent in the patients. Fifteen carriers of the Met-73 allele were found in six families of the familial combined hyperlipidemia group. Furthermore, six carriers of the Phe-334 allele were found in three families of the same group. In comparison to the controls the summarized odds ratio of 2.45 (95% confidence interval 0.89-6.71) barely missed the level of significance. The linkage between genotype and phenotype was incomplete. These results show an association of the missense mutations Val-73-Met and Leu-334-Phe as susceptibility alleles for combined forms of hyperlipidemia.
Restriction isotyping is a convenient method to perform Apo E genotyping. However, some experimental conditions can affect the restriction isotyping prpcedure and can lead to results prone to misinterpretation. It was evaluated whether the concentration of teraplate DNA or different methods of DNA Isolation have an influence on restriction isotyping. Reproducible results were achieved with 0.05l ng genomic DNA in a 100 PCR mixture. Using less DNA resulted in the appearance of non-specific bands. This could lead to a misinterpretation of the band pattern. An influence of different DNA Isolation procedures on restriction isotyping could be excluded. The appearance of the band pattern is influenced by the intensity of the bands which depends on the size of the fragment and on the formation of heteroduplexes and homoduplexes in the case of a heterozygote apo E genotype. A detailed Interpretation of the band patteras obtained by restriction isotyping of the apo E gene is given with regard to the intensity of the bands following ethidium bromide staining of the gel and UV photography.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.