Pyridinyl imidazole inhibitors, particularly SB203580, have been widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase (p38/HOG/SAP-KII) in a wide array of biological systems. Studies by this group and others have shown that SB203580 can have antiproliferative activity on cytokine-activated lymphocytes. However, we recently reported that the antiproliferative effects of SB203580 were unrelated to p38 MAP kinase activity. This present study now shows that SB203580 can inhibit the key cell cycle event of retinoblastoma protein phosphorylation in interleukin-2-stimulated T cells. Studies on the proximal regulator of this event, the phosphatidylinositol 3-kinase/protein kinase B (PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the phosphorylation and activation of PKB by inhibiting the PKB kinase, phosphoinositidedependent protein kinase 1. The concentrations of SB203580 required to block PKB phosphorylation (IC 50 3-5 M) are only approximately 10-fold higher than those required to inhibit p38 MAP kinase (IC 50 0.3-0.5 M). These data define a new activity for this drug and would suggest that extreme caution should be taken when interpreting data where SB203580 has been used at concentrations above 1-2 M. Interleukin-2 (IL-2)1 is a potent T cell growth factor that mediates its effects via a high affinity heterotrimeric receptor comprising ␣, ␤, and ␥ c subunits. Several intracellular signaling pathways are known to be activated by IL-2, including the p42/44 mitogen-activated protein kinase (MAP kinase, also known as ERK2/1), the p38 and p54 MAP kinases (also called stress kinases, or HOG and JNK, respectively), the phosphatidyl inositol 3Ј (PI) 3-kinase pathway and the Jak/STAT (signal transducer and activator of transcription) pathways. Our earlier studies using the MEK (mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase) inhibitor PD-098059 (1) and those of others (2, 3) have indicated that the p42/44 MAP kinase pathway is not required for IL-2-driven proliferation. In contrast, a pyridinyl imidazole inhibitor of p38 MAP kinase, SB203580, inhibited IL-2-driven T cell proliferation with an IC 50 of 3-5 M, suggesting a possible role for p38 MAP kinase in this process (4). Recently, we have further investigated the role of p38 MAP kinase in proliferation by mapping the subdomains of the IL-2 receptor ␤ chain involved in the activation of the kinase. As previously shown for p42/44 MAP kinase, activation of p38 and p54 MAP kinases required the acidic rich A region of the IL-2 receptor ␤ chain (5). However, the A region is not required for proliferation (2, 5), indicating that neither p38 MAP kinase nor p54 MAP kinase is essential for this function. Furthermore, CNI-1493 (6, 7), an inhibitor of p38 and p54 MAP kinase activation by IL-2 was unable to inhibit proliferation (5). Surprisingly, SB203580 was still able to inhibit proliferation in the absence of IL-2 stimulated p38 MAP kinase activation. It has already been reported that SB203580 does inhibit p54...

The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P 3 ) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1 A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P 3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC 50 ‫؍‬ 4.47 ؎ 1.88 nM, n ‫؍‬ 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC 50 ‫؍‬ 11.8 ؎ 4.2 nM, n ‫؍‬ 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2␣) with kinetics that closely resembled the accumulation of PI 3,4,5-P 3 . Moreover, this MCP-1-induced increase in PI3K-C2␣ activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2␣ rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2␣. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2␣ enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.Chemokines are a rapidly growing superfamily of 8 -10-kDa peptides that selectively attract and activate leukocyte populations (1, 2). Monocyte chemotactic peptide-1 (MCP-1) 1 (1) is a member of the CC chemokine family (1, 2), is a potent inducer of monocyte and CD45RO ϩ lymphocyte chemotaxis (3, 4), and also activates host defense mechanisms such as superoxide release (5). In vivo studies suggest that MCP-1 recruits monocytes to sites of inflammation in a variety of pathological conditions including atherosclerosis (6) and rheumatoid arthritis (7) as well as pulmonary fibrosis and granulomatous lung disease (8). MCP-1 has also been demonstrated to augment cytotoxic lymphocyte and natural killer cell activity in vitro, suggesting a novel role for chemokines as costimulators of T cell activation (9). Support fo...

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Aim To demonstrate quantitatively the range and extent of a clinical pharmacist's activities that can be delegated to pharmacy technicians without compromising patient care. Method All activities undertaken by the clinical pharmacist were evaluated and reassigned to the technicians if they met strict criteria. A job description for the technician was developed and approved by management and staff pharmacists. Data were collected and assessed prospectively for three staffing models: original model (1 pharmacist:30 beds, no technician); initial model (1 pharmacist:45 beds, 1 technician); and revised model (1 pharmacist:40 beds, 1 technician). The following key performance indicators were collected by clinical pharmacists before and after implementing the new models: number of clinical interventions recorded; number of medication history interviews completed; time lapsed after admission before a medication history interview is completed; and a surrogate indicator of pharmacy workload. Results The initial model showed a negative outcome in all areas and resulted in the model being revised. The revised model demonstrated that there was no negative difference in the key performance indicators compared with the original model. Conclusion Ward pharmacy technicians can assist pharmacists in achieving optimum patient care and quality use of medicines.

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.