Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells.

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor.

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.

The D-mannose-specific lectin from Gerardia savaglia blocks binding of human immunodeficiency virus type I to H9 cells and human lymphocytes in vitro.

The new D-mannose-specific lectin from Gerardia savaglia is shown to prevent infection of H9 cells with human immunodeficiency virus type 1 (HIV-1; strain HTLV-IIIB). At a concentration of 0.2 microM, complete protection was achieved. Even at a 50-fold higher concentration, this lectin is not toxic for the cells. Moreover, the lectin inhibits syncytium formation in the HTLV-IIIB/H9-Jurkat cell system to 100% at 0.2 microM. This effect was abolished by coaddition of D-mannose at a stoichiometric ratio of lectin to sugar of 1:500. The lectin-caused inhibition of syncytia formation was observed also in the HIV-1/human lymphocyte system. Perhaps more importantly, it is shown that the lectin reacts with the oligosaccharide side chains of the HIV-1 gp120 env molecule, which very likely can be classified to the high-mannose oligosaccharides. These data provide the basis for a rational screening for compounds interfering with gp120-CD4 interactions.

Differential modulation of host cell and HIV gene expression by combinations of avarol and AZT in vitro.

Human immunodeficiency virus type 1 (HIV-1) gene expression is modulated by some virus-encoded proteins, possibly acting at multiple levels of control, which are also known to be involved in the regulation of gene expression in uninfected cells (transcriptional, post-transcriptional, nucleocytoplasmic transport, and translational control). Two anti-HIV-1 drugs, Avarol and 3'-azido-3'-deoxythymidine, which inhibit viral replication by differential mechanisms, were used to study the role of cytoplasmic factors in independent regulation of host cell and viral gene expression. Both drugs were found to inhibit viral replication and synthesis of virus-encoded protein in a synergistic manner, while at cytostatic concentrations, both compounds act antagonistically. ATP-induced transport of viral messengers from isolated nuclei is enhanced by total cytosolic protein from HIV-1-infected cells; a strong increase of the nucleocytoplasmic transport of pol mRNA was measured and, to a lesser extent the transport of certain cellular mRNA (e.g. interleukin-2) was augmented, while the transport of other cellular mRNA (actin) was not affected at all.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: a target for potential therapeutic agents.

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.

Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region.

Previous studies revealed that antisense oligodeoxynucleotides to specific regions of the human immunodeficiency virus-1 (HIV-1) are potent inhibitors of replication of HIV-1 in vitro (Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarin, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4143-4146). We now report that antisense RNA, synthesized in vitro using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes targeted by antibodies specific for the T cell receptor molecule CD3 almost completely inhibited HIV-1 production. The viral env segment covered a part of exon II of HIV-1 tat gene. No anti-HIV activity could be detected with similarly targeted liposome-encapsulated sense env RNA or with pol RNA synthesized in either the sense or antisense orientations, or with env region antisense RNA free in solution, or encapsulated in liposomes in the absence of the targeting antibody. A semiquantitative evaluation revealed that 4000-7000 RNA molecules became cell-bound in targeted liposomes; the half-life of the intracellularly present hybridizable antisense env RNA was approximately 12 h. Western blots showed that antisense env RNA suppressed tat gene expression by approximately 90% and gp160 production by 100%. These data were confirmed by immunoprecipitation studies. Northern blots (using an env probe) demonstrated the existence of all major HIV RNA species (9.3-, 4.3-, and 2.0-kb mRNA) in HIV-infected cells treated with antisense env RNA although at a reduced level. We conclude that the antisense env RNA inhibited viral protein production at the translational level.

Single-dose pharmacokinetics of temocapril and temocapril diacid in subjects with varying degrees of renal impairment.

OBJECTIVE: The aim of this study was to determine the influence of renal impairment on the single-dose pharmacokinetics of temocapril and its pharmacologically active metabolite, temocapril diacid. METHODS: A single oral dose of 20 mg temocapril hydrochloride was given after an overnight fast to eight healthy (control) subjects (group A, n = 8) with a mean baseline creatinine clearance (CLCR) of 115.2 ml.min-1 and to three groups of patients with decreased renal function (mean CLCR 56.9 ml in group B, n = 8, 30.0 ml.min-1 in group C, n = 8 and 15.4 ml.min-1 in group D, n = 5). RESULTS: The mean peak concentration and median time to peak concentration for both temocapril and its diacid metabolite as well as the man area under the curve (AUC0-infinity) for temocapril did not differ significantly between groups. The mean AUC0-infinity for temocapril diacid increased only two- to threefold from group A to D. The mean terminal elimination half-life (t1/2) for temocapril diacid was prolonged in subjects with impaired renal function. However, prolongation of mean t1/2 and increase in AUC0-infinity did not parallel the decrease of mean renal clearance for temocapril diacid. CONCLUSION: The results suggest the existence of an alternative pathway in addition to the renal excretion of temocapril, e.g. via the bile. This pathway substantially contributes to the elimination of the active metabolite, temocapril diacid, in patients with decreased renal function. Nonetheless, to avoid any risks, the dose of temocapril hydrochloride in patients with moderate to severe renal impairment should be reduced.

Comparison of temocapril and atenolol in the long-term treatment of mild to moderate essential hypertension.

This 1-year, dose-titration, General Practitioner (GP) study compared efficacy, tolerability and safety of oral temocapril (10-40 mg once daily) with atenolol (25-100 mg once daily) in mild to moderate adult hypertensives (diastolic blood pressure (DBP) 95-114 mmHg). A 12-week dose-titration period, randomized 3:1 temocapril: atenolol, preceded 40 weeks long-term treatment. An intent-to-treat population of 472 patients was analysed for efficacy after 12 weeks dose-titration. Sitting DBP fell significantly (p < 0.001) in both groups, by mean +/- standard deviation (SD) 15.9 +/- 5.7 mmHg on temocapril and by 16.6 +/- 5.9 mmHg on atenolol. Therapeutic equivalence was demonstrated using the two one-sided t-tests procedure according to Schuirmann (equivalence interval [theta 1 - theta 2] < or = 5 mmHg). Responders (DBP < or = 90 mmHg) represented 89.9% of temocapril and 94.0% of atenolol patients. The lower doses were effective in 70.9% of temocapril patients (10 or 20 mg) and in 63.7% of atenolol patients (25 or 50 mg), these doses being continued after the dose titration period. No clinically relevant changes in haematological, biochemical and urinalysis variables occurred. Adverse events were few, largely unrelated to treatment and comparable between groups. In conclusion, temocapril and atenolol proved to be therapeutically equivalent antihypertensives.

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.

Identification of a cell surface-associated protein involved in mouse neural cell aggregation by means of antibodies against the sponge aggregation factor.

Polyclonal antibodies were raised against the purified aggregation factor (AF) from the sponge Geodia cydonium to elucidate possible immunological relationships between adhesion molecules of lower multicellular eukaryotic systems (sponges) and those of vertebrates. This anti-AF recognized a series of polypeptides associated with the AF, among them also a polypeptide with a Mr of 47,000 (p47). The formation of the antibody-p47 immunocomplexes could be prevented by adsorbing the anti-AF with a brain extract from DBA/2J mice. Moreover, this brain polypeptide inhibited the AF-mediated aggregation of sponge cells. Interestingly, the anti-AF recognized a p37 molecule in the brains of 2- to 3-day-old mice; no reaction could be traced using brain extracts from animals older than 2 months. The anti-AF failed to interact with polypeptides from mouse liver or spleen. By indirect immunofluorescence staining the p37 was found to be localized on the plasma membranes of brain cells. Moreover, Fab' fragments of the anti-AF inhibited aggregation of mouse brain cells. These data indicate that the sponge anti-AF recognizes a p37 molecule in mouse brain cells which is either directly or indirectly involved in brain cell aggregation.

Pharmacokinetics of temocapril and temocaprilat after 14 once daily oral doses of temocapril in hypertensive patients with varying degrees of renal impairment.

AIMS: The aim of this study was to determine the potential influence of renal impairment on the pharmacokinetics of temocapril and its pharmacologically active diacid metabolite, temocaprilat. METHODS: Non-compartmental pharmacokinetics were assessed in four groups of hypertensive patients (n=8 per group, four investigational centres) with normal (creatinine clearance determined via 24 h urine sampling, CL[CR], > or = 60 ml min-1) and impaired renal function (CL[CR] 40-59, 20-39, < 20 ml min-1) after 14 once daily oral doses of 10 mg temocapril hydrochloride. RESULTS: For temocapril, there were no statistically significant differences in median tmax or mean Cmax, AUC(SS), t1/2,Z, CL/F between the four groups. Renal clearance, CL(R), for temocapril showed a linear decreasing trend with decreasing CL(CR) [mean (s.d.): 32.2 (10.7) to 3.7 (3.0) ml min-1]. Steady-state for temocaprilat was reached on day 5. For temocaprilat, no statistically significant differences in mean Cmax or median tma were detected. With decreasing mean CL(CR), mean AUC(SS) for temocaprilat increased statistically significantly although only 2.4-fold [mean (s.d.): 2115 (565) to 4989 (2338) ng ml-1 h] and t1/2,Z was prolonged [mean (s.d.): 15.2 (1.2) to 20.0 (7.5) h]. CL(R) for temocaprilat showed a linear decreasing trend with decreasing CL(CR) [mean (s.d.): 20.2 (4.3) to 3.0 (1.8) ml min-1]. CONCLUSIONS: These results indicate that impaired renal function has only a limited effect on the pharmacokinetics of temocapril and its active metabolite, temocaprilat. This may be attributed to the dual, i.e. renal and biliary, elimination pathway of the drug.