MRG1 binds to the LIM domain of Lhx2 and may function as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expression.

Tissue-specific expression of the alpha-subunit gene of glycoprotein hormones involves an enhancer element designated the pituitary glycoprotein basal element, which interacts with the LIM homeodomain transcription factor, Lhx2. In the present studies we have explored the function of the LIM domain of Lhx2 in stimulating alpha-subunit transcription. When fused to the GAL4 DNA-binding domain, the LIM domain of Lhx2 was shown to contain a transcriptional activation domain. Furthermore, in the context of an alpha-subunit reporter gene in which a GAL4-binding site replaced the pituitary glycoprotein basal element, the LIM domain enhanced both basal and Ras-mediated transcription. In addition, a synergistic response to Ras activation was observed when the Lhx2 LIM domain and the transactivation domain of Elk1 are directed to a minimal reporter gene. A yeast two-hybrid screen identified the recently described melanocyte-specific gene-related gene 1 (MRG1) as an Lhx2 LIM-interacting protein. MRG1 was shown to bind Lhx2 in vitro, and a co-immunoprecipitation assay provided evidence that endogenous MRG1 forms a complex with Lhx2 in alphaT3-1 cells. Expression of MRG1 in alphaT3-1 cells enhanced alpha-subunit reporter gene activity. MRG1 was also shown to bind in vitro to the TATA-binding protein and the transcriptional coactivator, p300. These data suggest a model in which the Lhx2 LIM domain activates transcription through interaction with MRG1 leading to recruitment of p300/CBP and the TATA-binding protein.

Regulation of glycoprotein hormone alpha-subunit gene expression.

The appropriate, regulated expression of the glycoprotein hormone subunit genes is required to enable the biosynthesis of luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, and chorionic gonadotropin. We have focused our attention on mechanisms mediating regulated transcription of the common alpha-subunit gene. Our studies have examined both the signaling mechanisms and the DNA elements and transcription factors that are important for alpha-subunit expression. Our initial efforts involved characterization of DNA elements of the alpha-subunit gene important for basal and GnRH-stimulated expression. Clustered point mutation analysis identified two different, unrelated sequences that play a role in alpha-subunit transcription. When tested as multiple copies on a minimal promoter, one of these elements was sufficient to permit a response to GnRH, while the other enhanced basal expression. Therefore, we designated these DNA elements as the GnRH-response element (GnRH-RE) and the pituitary glycoprotein hormone basal element (PGBE), respectively. The GnRH-RE contains a consensus binding site for the Ets family of transcription factors. As several Ets factors have been shown to mediate transcriptional responses to the mitogen-activated protein kinase (MAPK) pathway, we investigated the possibility that GnRH effects on alpha-subunit transcription may involve the MAPK cascade. We found that GnRH can indeed activate MAPK and that MAPK activation is sufficient and necessary for transcriptional activation of the alpha-subunit gene. Efforts to further characterize proteins that interact with the PGBE led to the cloning of a LIM-homeodomain transcription factor designated LH-2. Recombinant LH-2 selectively binds to the PGBE in vitro. Transfection experiments have shown that an expression vector for LH-2 can activate the alpha-subunit promoter in heterologous cells. LH-2 appears to be a component of the endogenous factors that bind to the PGBE. Thus, LH-2 appears to be an excellent candidate as a factor responsible for basal expression of the alpha-subunit gene. Overall, these studies have contributed to identification of molecular components important for regulated expression of the glycoprotein hormone alpha-subunit gene.

Discharging patients with prescriptions instead of medications: sequelae in a teaching hospital.

OBJECTIVE: This study measures the incidence of discrepancies among written prescriptions, medication regimens transcribed onto patient discharge instruction sheets (DCIs), and labels on medications dispensed by community pharmacies after discharge of patients from an academic medical center. METHODS: During a 2-month study period, we collected copies of prescriptions and DCIs. We also called care givers after discharge and asked them to read the medication labels that were filled from discharge prescriptions. Care givers were also asked whether they received instruction from community pharmacies. RESULTS: Data were collected on 335 prescriptions for 192 patients. Differences among the prescriptions, DCIs, and medication labels were found for 40 (12%) of the medications prescribed at discharge, representing 19% of the patients studied. Nineteen prescriptions had prescriber errors in dosing frequencies or dosage formulations. Three prescriptions were filled with different medication concentrations or strengths than requested. Prescriptions were altered by the community pharmacists for unexplained reasons in 6 cases, whereas the DCIs and original prescriptions differed in 12 cases. Only 44% of families were counseled about proper medication administration by their pharmacists. CONCLUSIONS: A potential for medication errors exists when pediatric patients are discharged with unfilled prescriptions. The potential may be worsened when discharge instructions are created from a prescription rather than from the label of a dispensed medication. Educational and risk-management efforts should emphasize the importance of writing complete, legible prescriptions and consulting appropriate reference materials to ensure that dose formulations and guidelines are accurate. Whenever possible, prescriptions should be filled before patients are discharged, so that the dispensed medications can be reviewed, and health care providers can provide accurate discharge instructions.

Cefotaxime and ceftriaxone use evaluation in pediatrics. Considerations of cost effectiveness.

In 1993, there was a change from ceftriaxone to cefotaxime in the inpatient pediatric division of the Johns Hopkins Hospital. The annual cost savings resulting from this change were estimated. The educational efforts of the pediatric division pharmacists resulted in an increase in appropriate drug selection from 55% to 93%. The estimated annual cost saving was $18,618.

Chlorinated phenyl azides as photolabeling reagents. Synthesis of an ortho,ortho'-dichlorinated arylazido PCP receptor ligand.

The enhanced photolabeling properties of chlorinated phenyl azides are demonstrated by the synthesis and photolysis of methyl 4-azido-2,3,5,6-tetrachlorobenzoate (3) and methyl 4-azido-3,5-dichlorobenzoate (4). Photolysis of azide 3 in 1 M diethylamine/cyclohexane as the trapping medium gave 34% NH-insertion product. Similar photolysis of azide 4 gave 35% NH insertion product. These results demonstrate that chlorinated phenyl azides are significantly better at undergoing NH insertion than nonhalogenated analogs and suggest that improvement of existing aryl azide-based photolabels might be achieved by introduction of chlorine atoms on either side of the azide group. As an application, 3-azido-2,4-dichloro-10,5-(iminomethano)-10,11-dihydro-5H- dibenzo[a,d]cycloheptene (19), an analog of the potent PCP receptor ligand IDDC (14), was synthesized and its affinity for the PCP receptor was determined to be 6.3 +/- 0.7 microM (IC50 against [3H]MK801).