Relative levels of inhibition of p24 gene expression by different 20-mer antisense oligonucleotide sequences targeting nucleotides + 1129 to + 1268 of the HIV-1 gag genome: an analysis of mechanism.

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1 gag gene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of the gag gene, at positions that 5' or 3' flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

Antiviral activity and protection of cells against human immunodeficiency virus type-1 using an antisense oligodeoxyribonucleotide phosphorothioate complementary to the 5'-LTR region of the viral genome.

A COS-like monkey kidney cell line stably transfected with the plasmids pCMVgagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a model for assessing the effectiveness of antisense (AS) constructs, A 20-mer oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long terminal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blot analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-microM range. When lipofectin was used as a delivery vehicle, a markedly increased potentiation of the AS activity of the sequence was observed at a lower concentration (0.1 microM), following a 24-h preincubation. The AS construct specifically inhibited intracellular p24 production in chronically HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, resulting in a 15-fold inhibitory effect relative to a similar sequence thiolated at only seven single-base positions. A concentration-dependent attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretreated HIV-1-infected phytohemagglutinin A-stimulated human cord blood mononuclear cells. Incubation of a HIV-1-infected lymphoid cell line with AS sequence resulted in a marked reduction in syncytium formation, and therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell growth rate and colony-forming ability assays. The AS oligo described in this report is a useful new tool for the molecular analysis of HIV-1 gene expression and proliferation, and may have potential as a therapeutic agent.

Inhibition of human immunodeficiency virus type 1 (HIV) gag gene expression by an antisense oligodeoxynucleotide phosphorothioate.

We have employed a cos-like monkey kidney cell line (B4.14) transfected with plasmids pCMVgag-pol-rre-r (containing the HIV gag and pol genes) and pCMVrev (containing the HIV rev gene), as a model to investigate whether antisense constructs could interfere with specific HIV gene and protein expression. We utilized an antisense construct (GP12A) directed against a non-regulatory region of the HIV genome, to transfect cells that expressed the above-mentioned HIV genes. Our results show that GP12A was able to attenuate levels of relevant HIV mRNA and gag proteins in the absence of cytotoxic effects.

Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome.

Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A.

Boswellic acids: novel, specific, nonredox inhibitors of 5-lipoxygenase.

Isomers (alpha- and beta-) of boswellic acids (BAs), 11-keto-beta-BA and their acetyl derivatives were isolated from the gum resin of Boswellia serrata. BA and derivatives concentration dependently decreased the formation of leukotriene B4 from endogenous arachidonic acid in rat peritoneal neutrophils. Among the BAs, acetyl-11-keto-beta-BA induced the most pronounced inhibition of 5-lipoxygenase (5-LO) product formation with an IC50 of 1.5 microM. In contrast to the redox type 5-LO inhibitor nordihydroguaiaretic acid, BA in concentrations up to 400 microM did not impair the cyclooxygenase and 12-lipoxygenase in isolated human platelets and the peroxidation of arachidonic acid by Fe-ascorbate. The data strongly suggest that BAs are specific, nonreducing-type inhibitors of the 5-LO product formation either interacting directly with the 5-LO or blocking its translocation.

Calmodulin- and Ca2(+)-insensitive fatty acid methyltransferase from RINm5F cells. Inhibition by trifluoperazine and W7.

1. Methylation of endogenous lipids by homogenates of rat insulinoma cells was studied. 2. 3H-methyl groups (38 pmol/mg protein per 10 min) from [3H-methyl]S-adenosyl-L-methionine were incorporated into endogenous lipids, mainly (greater than 80%) into the neutral lipid fraction. 3. The reaction was sensitive to heat, was almost abolished by S-adenosyl-L-homocysteine, but insensitive to the addition of EGTA (5 mM), Ca2+ (5-100 microM) and/or calmodulin (15 microns). 4. At concentrations relevant for calmodulin antagonistic activity strong inhibition by W7 and trifluoperazine (25-100 microM each), but not by CGS 9343B (10 microM), was observed. 5. Calmodulin antagonists of phenothiazine- and sulfonamide-type appear to block the fatty acid methyltransferase in a way unrelated to calmodulin.

Potentiation of forskolin-induced increase of cAMP by diamide and N-ethylmaleimide in rat pancreatic islets.

In isolated rat pancreatic islets, the effect of diamide and N-ethylmaleimide (NEM) on forskolin- as well as on glucagon-induced elevation of cAMP was studied. Forskolin and glucagon increased cAMP levels in batch-incubated islets. Diamide and NEM further augmented forskolin-induced increase of cAMP levels, whereas glucagon-stimulated elevation of cAMP was not affected. From our data it is likely that under the conditions of the present study, the thiols related to the Ns-protein and the catalytic unit are insensitive to oxidation and alkylation. The potentiation of forskolin-induced cAMP production in intact islet cells by diamide and NEM may be due to inactivation of the thiols related to the Ni-protein.

Thiol reagents (diamide and N-ethylmaleimide) inhibit increase in cAMP in response to glucose and abolish the clonidine-mediated attenuation of glucagon-induced cAMP formation in isolated rat pancreatic islets.

In freshly collagenase-isolated rat pancreatic islets and in islets cultured for 72 hours, the effects of thiol reagents on glucagon (5 micrograms/ml) and/or glucose (16.7 mM)-mediated increases in cAMP formation as well as on clonidine (10 microM)-induced inhibition of these actions were studied. In freshly isolated islets and to a more pronounced degree in islets cultured for 72 hours glucagon (5 micrograms/ml) increased the cAMP content above the basal value. Clonidine (0.1-100 microM) had no significant effect on the basal cAMP formation, but inhibited the glucagon-mediated effect. The thiol reagents diamide (10-100 microM) and NEM affected neither the basal nor the glucagon-mediated effect, but abolished the inhibitory action of clonidine on cAMP formation. In freshly isolated islets, high glucose concentrations (8.3-16.7 mM) increased the cAMP formation. Diamide (100 microM) and NEM (100 microM) attenuated the stimulatory effect of 16.7 mM glucose. It is suggested that these selective effects of the thiol reagents on glucagon-mediated increase in cAMP formation in the presence of substimulatory concentration of glucose may be due to the differences in the sensitivity of the sulfhydryl groups of the G-proteins to thiol reagents i.e. Gi or proteins closely related to Gi being more sensitive than Gs. The data further suggest that glucose acts on the cAMP cascade at a step distinct from Rs. Since both glucose and glucagon effects were influenced by the addition of clonidine, it is possible to interpret the data as indicating that the effects of both stimulators eventually converge at some common step in the adenylate cyclase cascade.

Effects of propylthiouracil and methylthiouracil on cyclic AMP and ion movements in rat pancreatic islets.

Propylthiouracil and methylthiouracil have been shown to potentiate glucose-induced insulin secretion from rat pancreatic islets: the effect of methylthiouracil being less pronounced than that of propylthiouracil. In this study the effects of these substances on cAMP levels, 86Rb+ efflux, 45Ca2+ net uptake, and 45Ca2+ efflux were tested in isolated rat islets in order to obtain information on their possible mechanism of action. Propylthiouracil and to a lesser extent methylthiouracil increased islet cyclic AMP in a concentration-related manner. Maximum increases at the highest concentrations tested were 261% and 190% respectively. In the presence of 3 mM glucose propylthiouracil and methylthiouracil led to a decrease in the 86Rb efflux rate. With 5.6 mM glucose, both thiourea derivatives produced an increase in the 86Rb+ efflux rate which was independent of the presence or absence of calcium in the medium. Propylthiouracil and methylthiouracil augmented the 45Ca2+ efflux rate in the presence as well as in the absence of external calcium at various glucose concentrations. Propylthiouracil did not change, and methylthiouracil only slightly augmented, 45Ca2+ net uptake into the isolated islets. It is suggested that the synergistic effect of propylthiouracil and methylthiouracil on glucose-induced insulin release is at least in part due to an increase in islet cAMP levels. Whether the two substances have additional direct effects on ionic fluxes which contribute to their insulinotropic action or whether the observed changes in ion movements are secondary to the elevation of cAMP levels remains to be unclear and needs further investigation.