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.

A role for prostacyclin in bruising symptomatology.

The relationship between bleeding and bruising and the production of prostacyclin and thromboxane was assessed in children who were to have a tonsillectomy and/or an adenoidectomy. Eicosanoids in the blood oozing from the bleeding time incision were measured and correlated with the reported frequency of bruising and epistaxis. A striking association (P = .0003) between prostacyclin production and the frequency of bruising was found; children reporting bleeding at least biweekly had the highest prostacyclin synthesis. Successively lower levels of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha, were found in children reporting less frequent bruising. Prostacyclin production in bleeding time blood was also correlated inversely with systolic blood pressure and hemoglobin level, although neither of these variables could explain the association between prostacyclin production and bruising. There was no correlation between thromboxane formation, systolic blood pressure, hemoglobin level, age, or bleeding time and the frequency of bruising. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was correlated inversely with the length of the bleeding time (P = .016). It is concluded that vascular prostacyclin production may have a role in bruising symptomatology. It is suggested that prostacyclin formed at the injured vessel surface collects within the first few seconds after injury inside the tissue space at the site of the bruise and, by influencing the formation of the platelet/fibrin plug and/or the leakage of blood from the vessels, plays a significant role in modifying the development of bruising.

Studies in patients with bleeding disorders show that platelet-vessel interaction is important for thromboxane formation in bleeding time wounds.

The production of thomboxane B2, the primary metabolite of thromboxane A2, and 6-keto prostaglandin F1 alpha, the primary metabolite of prostacyclin, were measured in response to a standardized vascular injury, the bleeding time, in patients with von Willebrand's disease and in patients with platelet function defects. Compared to controls, thromboxane B2 levels in bleeding time blood were significantly lower in subjects with von Willebrand's disease. In patients with platelet function defects associated with a deficient response to thromboxane A2, thromboxane B2 production in bleeding time blood was similar to controls. In subjects with other platelet function defects, thromboxane production was significantly lower than normal. 6-keto PGF1 alpha production in bleeding time blood was not significantly different in patients compared to controls. The results suggest that bleeding time thromboxane production is influenced by the extent of platelet-vessel interaction.

The effect of exercise on bleeding time and local production of prostacyclin and thromboxane.

Exercise at a heart rate corresponding to 30% VO2max for 15 min was associated with an increase in the volume of bleeding time blood from a mean of 133 microliters before exercise to a mean of 218 microliters during and immediately after the exercise. There was similarly an increase in thromboxane B2 production from 6.40 nmol.l-1 before to 11.50 nmol.l-1. Most subjects also showed an increase in the length of the bleeding time and in the production of bleeding time 6-keto-PGF1 alpha. The extent of increase in the bleeding time and in production of 6-keto-PGF1 alpha was quite variable, with subjects showing the largest increases in bleeding time also demonstrating the greatest increases in 6-keto-PGF1 alpha (r = 0.76, P = 0.004). The ingestion of aspirin before exercise markedly inhibited basal bleeding time thromboxane B2 production and blocked the exercise-associated increments in thromboxane B2 and 6-keto-PGF1 alpha production. While the aspirin itself increased the length of the bleeding time, there was not any further increase associated with exercise. In contrast to the effects of acute short-term exercise, long-distance running was associated with a significant decrease in bleeding time, but no change in bleeding time blood volume, bleeding time thromboxane B2, or bleeding time 6-keto-PGF1 alpha. The results show that acute low-level exercise can be associated with significant changes in the volume of blood oozing from a bleeding time incision and in the amount of thromboxane production stimulated at the incisional site. Following exhaustive exercise of long duration, the above changes are no longer seen.

Chlorpromazine-induced decrease of red cell aggregability.

Starting from the assumption that binding of chlorpromazine to the erythrocyte membrane might influence th rheology of these cells, the authors studied erythrocyte aggregability and deformability by means of a filtrability test in 42 neurotics and 16 patients with personality disorders, all males, aged 20 to 27 years, clinically healthy from the somatic view point. Determinations were carried out before and 30 min after a single i.m. dose of 1 mg/kg b.w. chlorpromazine (14 patients) and before and after a 5-day oral treatment with 3 mg/day haloperidol (11 patients) or 75 mg/day chlorpromazine (33 patients). Chlorpromazine induced a significant decrease of erythrocyte aggregability after 5 days of oral treatment (p less than 0.02) the effect being more marked in the patients with relatively high initial values (p less than 0.01). The mechanism of this phenomenon and its possible implications of ESR determination and in the therapy of occlusive vascular diseases are discussed.