1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.

The effect of the novel, naturally occurring nucleotide cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid CD4+ Jurkat cell line was studied. When exposed to 50 W WM c-di-GMP, Jurkat cells exhibited a markedly elevated expression of the CD4 receptor of up to 6.3-fold over controls. C-di-GMP also causes blockage of the cell cycle at the S-phase, characterized by increased cellular thymidine uptake, reduction in G2/M-phase cells, increase in S-phase cells and decreased cell division. Additionally c-di-GMP naturally enters these cells and binds irreversibly to the P21 r s protein. The effects described appear to be unique for c-di-GMP.z 1999 Federation of European Biochemical Societies.

Several studies in human and experimental models indicate the existence of a partial relationship between essential hypertension (EH) and the immune system. In this study, cellular immune functions were investigated in 13 patients with untreated and uncomplicated essential hypertension (EHP) and in 10 of their offspring (EHO) and compared to 13 age- and sex-matched normotensive controls (NC) and 10 of their offspring (NCO). The total number of T cells and T cell subsets were similar in all groups examined. In the EHP, basal lymphocyte transformation without lectins was significantly lower (1,126 ± 261 cpm of [³H]-thymidine uptake) than in the NC (3,223 ± 736, p < 0.01); the response to both phytohemagglutinin (PHA) and concanavalin A (ConA) revealed reduced [³H]-thymidine uptake as compared with NC (21,890 ± 5,432 compared to 64,574 ± 9,723 for PHA and 10,488 ± 2,621 compared to 37,334 ± 8,148 for ConA, respectively, p < 0.01). However, the ability to proliferate as a response to lectins was normal. This was leading to a normal stimulation index in both groups. In the EHO, non-significant decrease in basal transformation and reduced uptake with PHA (49,537 ± 7,478) versus NCO (69,911 ± 7,254) and NC (64,574 ± 9,723) were found. These findings suggest that the proliferative response of T lymphocytes is partially suppressed in EH.

T cell function tests were performed in 6 patients with uncomplicated essential hypertension (EH) before and after treatment with captopril, an angiotensin-converting enzyme inhibitor. Total T and T cell subsets were within normal range and were not affected by the drug. The response of PBL to lectin stimulation was significantly impaired. While the stimulation index (SI) was almost normal when washed PBL were used (32.2 ± 6.90 with PHA and 20.17 ± 4.1 with Con A) or after their incubation with serum taken from normal subjects (40.42 ± 10.9 and 15.53 ± 3.4, respectively), autologous serum significantly reduced the SI (15.04 ± 5.9 with PHA, p < 0.05 and 6.68 ± 1.45 with Con A, p < 0.005). While captopril seemed to supress the SI of washed PBL after 1 week of treatment, it enhanced the SI from 32.21 ± 6.91 to 55.32 ± 10.76 for PHA and from 20.17 ± 4.13 to 30.63 ± 5.41 for Con A (p < 0.001). This effect was more obvious when the stimulations were performed with normal serum (from 40.42 ± 10.9 to 96.47 ± 17.51 for PTH and from 15.53 ± 3.43 to 40.02 ± 8.0 for Con A, (p < 0.001). These results confirm previous reports indicating that the cellular immune response is impaired in EH. It seems that an inhibitory factor found in the serum of these patients is responsible for this impairment. Our findings may suggest that this factor may be angiotensin II.