Mouse Models of Gonadotrope Development.

The pituitary gonadotrope is central to reproductive function. Gonadotropes develop in a systematic process dependent on signaling factors secreted from surrounding tissues and those produced within the pituitary gland itself. These signaling pathways are important for stimulating specific transcription factors that ultimately regulate the expression of genes and define gonadotrope identity. Proper gonadotrope development and ultimately gonadotrope function are essential for normal sexual maturation and fertility. Understanding the mechanisms governing differentiation programs of gonadotropes is important to improve treatment and molecular diagnoses for patients with gonadotrope abnormalities. Much of what is known about gonadotrope development has been elucidated from mouse models in which important factors contributing to gonadotrope development and function have been deleted, ectopically expressed, or modified. This chapter will focus on many of these mouse models and their contribution to our current understanding of gonadotrope development.

Molecular mechanisms of pituitary organogenesis: In search of novel regulatory genes.

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or non-syndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

In vitro characterization and pharmacokinetics of dapagliflozin (BMS-512148), a potent sodium-glucose cotransporter type II inhibitor, in animals and humans.

(2S,3R,4R,5S,6R)-2-(3-(4-Ethoxybenzyl)-4-chlorophenyl)-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol (dapagliflozin; BMS-512148) is a potent sodium-glucose cotransporter type II inhibitor in animals and humans and is currently under development for the treatment of type 2 diabetes. The preclinical characterization of dapagliflozin, to allow compound selection and prediction of pharmacological and dispositional behavior in the clinic, involved Caco-2 cell permeability studies, cytochrome P450 (P450) inhibition and induction studies, P450 reaction phenotyping, metabolite identification in hepatocytes, and pharmacokinetics in rats, dogs, and monkeys. Dapagliflozin was found to have good permeability across Caco-2 cell membranes. It was found to be a substrate for P-glycoprotein (P-gp) but not a significant P-gp inhibitor. Dapagliflozin was not found to be an inhibitor or an inducer of human P450 enzymes. The in vitro metabolic profiles of dapagliflozin after incubation with hepatocytes from mice, rats, dogs, monkeys, and humans were qualitatively similar. Rat hepatocyte incubations showed the highest turnover, and dapagliflozin was most stable in human hepatocytes. Prominent in vitro metabolic pathways observed were glucuronidation, hydroxylation, and O-deethylation. Pharmacokinetic parameters for dapagliflozin in preclinical species revealed a compound with adequate oral exposure, clearance, and elimination half-life, consistent with the potential for single daily dosing in humans. The pharmacokinetics in humans after a single dose of 50 mg of [(14)C]dapagliflozin showed good exposure, low clearance, adequate half-life, and no metabolites with significant pharmacological activity or toxicological concern.

Is gonadotrope expression of the gonadotropin releasing hormone receptor gene mediated by autocrine/paracrine stimulation of an activin response element?

Expression of the FSHbeta subunit and GnRH receptor (GnRHR) genes in gonadotropes is stimulated by activin. We sought to identify the cis-acting element(s) in the murine GnRHR gene promoter which confer activin responsiveness. We established that 600 bp of 5'flanking sequence from the murine GnRHR gene were sufficient to confer activin responsiveness in the gonadotrope-derived alphaT3-1 cell line. Since alphaT3-1 cells, like gonadotropes, secrete activin, we examined the ability of follistatin, an activin binding protein, to block the activin response. Increasing concentrations of follistatin from 0 to 100 ng/ml resulted in a dose dependent decrease in activity of the -600 promoter. Contained within this region are three elements important for expression in alphaT3-1 cells: a Steroidogenic Factor-1 binding site (SF-1), an Activator Protein-1(AP-1) element, and an element termed the GnRH receptor activating sequence or GRAS. A block mutation of GRAS inhibited the ability of the promoter to respond to follistatin. A more refined analysis using a series of two-bp mutations which scan GRAS and flanking sequence revealed exact convergence of GRAS with activin/follistatin responsiveness. Finally, a construct consisting of 3 copies of GRAS placed upstream of a heterologous minimal promoter (3xGRAS-PRL-LUC) was responsive to both activin stimulation and follistatin inhibition in alphaT3-1 cells. Thus, autocrine/paracrine stimulation of gonadotropes by activin illustrates a unique mechanism for cell-specific gene expression.

Synthesis and evaluation of inhibitors of bacterial D-alanine:D-alanine ligases.

BACKGROUND: D-Alanine:D-alanine ligase is essential for bacterial cell wall synthesis, assembling one of the subunits used for peptidoglycan crosslinking. The resulting aminoacyl-D-Ala-D-Ala strand is the Achilles' heel of vancomycin-susceptible bacteria; binding of vancomycin to this sequence interferes with crosslinking and blocks cell-wall synthesis. A mutant enzyme (VanA) from vancomycin-resistant Enterococcus faecium has been found to incorporate alpha-hydroxy acids at the terminal site instead of D-Ala; the resulting depsipeptides do not bind vancomycin, yet function in the crosslinking reaction. To investigate the binding specificity of these ligases, we examined their inhibition by a series of substrate analogs. RESULTS: Phosphinate and phosphonate dipeptide analogs (which, after phosphorylation by the enzyme, mimic intermediates in the ligation reaction) were prepared and evaluated as reversible inhibitors of the wild-type ligases DdlA and DdlB from Escherichia coli and of the mutant enzyme VanA. Ki values were calculated for the first stage of inhibitor binding according to a mechanism in which inhibitor competes with D-Ala for both substrate binding sites. DdlA is potently inhibited by phosphinates but not by phosphonates, while DdlB and VanA show little discrimination; both series of compounds inhibit DdlB strongly and VanA weakly. CONCLUSIONS: VanA has greatly reduced affinity for all the ligands studied. The relative affinities of the inhibitors in the reversible binding step are not, however, consistent with the substrate specificities of the enzymes. We propose a mechanism in which proton transfer from the attacking nucleophile to the departing phosphate occurs directly, without intervention of the enzyme.

Shielding technique for small ports in the superficial and orthovoltage range.

A simple reproducible technique for construction of shields for treatment with orthovoltage and superficial equipment is described. This technique also provides immobilization of treatment areas when treatment cones are utilized.