Publications

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.

Staphylococcus aureus clinical isolates obtained from patients who were inmates of the San Francisco County jail system showed an increase in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) from 29%, in 1997, to 74%, in 2002; 91% of the MRSA isolates carried staphylococcal chromosomal cassette mec (SCCmec) type IV. Pulsed field gel electrophoresis and multilocus sequence typing demonstrated 2 major clonal groups. One of these clonal groups is genetically indistinguishable from the strain responsible for an outbreak of MRSA in the Los Angeles County jail system in 2002.

Concern about the effects of anesthesia on physiological measurements led us to develop methodology to assess left ventricular (LV) pressure in conscious mice. Polyethylene-50 tubing filled with heparinized saline was implanted in the LV cavity through its apex via an abdominal approach and exteriorized to the back of the animal. This surgery was done under anesthesia with either an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) (K+X) in 11 mice or isoflurane (ISF; 1.5 vol%) by inhalation in 14 mice. Postoperatively, mice were trained daily to lie quietly head first in a plastic cone. LV pressure, the first derivative of LV pressure (dP/dt), and heart rate (HR) in the conscious state were compared between the two groups at 3 days and 1 wk after recovery from surgery using a 1.4-Fr Millar catheter inserted into the LV through the tubing, with the mice lying quietly in the plastic cone. Acutely during anesthesia, K+X decreased HR (from 698 to 298 beats/min), LV systolic pressure (from 107 to 65 mmHg), and maximal dP/dt (dP/dt(max)) (from 15,724 to 4,445 mmHg/s), all P < 0.01. Similar but less marked negative chronotropic and inotropic effects were seen with ISF. HR and dP/dt(max) were decreased significantly in K+X mice 3 days after surgery compared with those anesthetized with ISF (655 vs. 711 beats/min, P < 0.05; 14,448 vs. 18,048 mmHg/s, P < 0.001) but increased to the same level as in ISF mice 1 wk after surgery. In ISF mice, recovery of function occurred rapidly and there were no differences in LV variables between 3 days and 1 wk. LV pressure and dP/dt can be measured in conscious mice with a micromanometer catheter inserted through tubing implanted permanently in the LV apex. Anesthesia with either K+X or, to a lesser extent, ISF, depressed LV function acutely. This depression of function persisted for 3 days after surgery with K+X (but not ISF) and did not recover completely until 1 wk postanesthesia.

Translocation of the bcl-2 gene to the immunoglobulin heavy chain gene is the most common alteration in follicular lymphoma. The result is the deregulated expression of bcl-2 and increased resistance to cell death. Regulation of the immunoglobulin heavy chain gene is controlled in part by four DNase I-hypersensitive regions located 3' of the gene. Here, we show that these four enhancer regions also contribute to bcl-2 up-regulation in t(14;18) cells. The enhancers are able to individually or in combination activate bcl-2 promoter activity. The HS4 enhancer region was found to impart the largest positive effect on the bcl-2 promoter, activating it by 6-fold, whereas addition of the HS1,2 region with HS4 increased promoter activity by approximately 9-fold. Nuclear factor kappaB binding sites were shown to be primarily responsible for the positive activity contributed by the HS1,2 and HS4 regions, and we observed the in vivo interaction of these factors with the human immunoglobulin heavy chain gene enhancer regions in t(14;18) cells. In addition, two Sp1 binding sites in HS4 were also found to positively influence bcl-2 activity, and Sp1 was observed to interact with the human HS4 enhancer in vivo. These results suggest that the interactions of the nuclear factor kappaB and Sp1 transcription factors with the immunoglobulin heavy chain enhancer region are important for bcl-2 deregulation in t(14;18) cells.