Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes.

Cyclins and cyclin-dependent kinases are critically involved in controlling cell cycle progression in virtually all cells. The recent identification of candidate G1 cyclins in mammalian cells has been a major advance in this field, but the exact functions of these cyclins are unknown. The expression of three D-type cyclins (D1, D2, and D3) was investigated in primary human T lymphocytes as these cells were induced to leave G0, traverse G1, and enter S phase by T cell-specific mitogens. G0 phase T cells expressed low levels of cyclin D2, but not cyclin D3. Treatment of these cells with phytohemagglutinin and 12-O-tetradecanoylphorbol-13-acetate in the presence of fetal calf serum resulted in rapid induction of cyclin D2 RNA in early G1 and slower induction of cyclin D3 in late G1. Cyclin D1 was not detected in T cells under any condition tested. Treatment of T cells with hydroxyurea to arrest cells at G1/S did not block induction of either D2 or D3. However, arrest of cells in mid G1 with deferoxamine blocked D3 expression without affecting D2. Cyclosporin A blocked the induction of both cyclin D2 and D3. Polyclonal antisera were prepared in rabbits against both cyclin D2 and cyclin D3 glutathione S-transferase fusion proteins and used to examine cyclin D2 and D3 proteins in [35S]methionine-labeled T cells. Protein levels were found to correlate closely with RNA levels for both cyclins. No detectable histone H1 kinase activity could be precipitated with either cyclin. However, several cellular proteins were observed to coprecipitate with the cyclins, including several proteins that were observed to associate only with D3. These results indicate that striking differences exist in the induction and regulation of two candidate G1 cyclins in human T cells and suggest that these cyclins could participate in multiple cell cycle checkpoints during G0, G1, or S phase.

Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties.

An expression vector was modified to permit the rapid synthesis of purified, 32P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen lambda gt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB "pocket" is known to bind, directly or indirectly, to the cellular transcription factor, E2F, implicated in cell growth control. We have found that RBAP-1 copurifies with E2F, interacts specifically with the adenovirus E4 ORF 6/7 protein, binds specifically and directly to a known E2F DNA recognition sequence, and contains a functional tranasactivation domain. Therefore, RBAP-1 is a species of E2F and can bind specifically to the RB pocket.

The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression.

The retinoblastoma-susceptibility gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. We characterized RB phosphorylation after mitogenic stimulation of primary human T lymphocytes, initially arrested in the G0 state. RB is phosphorylated in at least three steps when T cells are driven into the cell cycle. The first event occurs during mid G1 phase, the second during S phase, and the third in G2/M. Tryptic phosphopeptide mapping indicates that the different phosphorylation events occur, at least in part, on different residues in RB. Given the known relationship of the RB phosphorylation state to function, it is possible that RB regulates growth at multiple points in the cell cycle.

[Antibiotic therapy protocol using ceftazidime 3g/day alone or in combination with vancomycin or amikacin. In febrile episodes in neutropenic patients]. / Protocole d'antibiothérapie utilisant la ceftazidime à la dose de 3 g/jour seule ou en association avec la vancomycine ou l'amikacine. Lors des épisodes fébriles chez le neutropénique.

In a preliminary study of 21 febrile episodes in neutropenic patients ceftazidime used as empirical treatment in doses of 3 grams per day succeeded in controlling fever in 74 per cent of the cases. Laboratory studies performed in patients with Gram-negative septicaemia showed clinically effective plasma concentrations of the antibiotic. A trial of ceftazidime (3 g/day) administered alone or combined with amikacin or vancomycin is currently in progress in two medical centres. No statistically significant conclusions could be reached from an intermediate study.

[Chronic lymphocytic leukemia, erythroblastopenia, thymolipoma]. / Leucémie lymphoïde chronique, érythroblastopénie, thymolipome.

The association of B cell chronic lymphocytic leukemia and pure red cell aplasia in a 42 year-old patient led to the discovery of a thymic enlargement. After six weeks treatment including steroids, cyclophosphamide and three courses of plasma exchange without improvement, surgical thymectomy was followed by a reticulocytosis and remission of the red cell aplasia. The tumor was a thymolipoma. Characteristics of this are thymic tumor, his connections with pure red cell aplasia and in vitro differentiation of erythroid progenitors are discussed.