Publications

Heterozygous coding mutations in the melanocortin 4 receptor (MC4R) are implicated in 1 to 6% of early onset or severe adult obesity cases. To better address the problem of the genotype:phenotype relationship within this specific form of obesity, we systematically studied the functional characteristics of 50 different obesity-associated MC4R mutations. Structure modeling of MC4R indicates that obesity-associated MC4R mutations are not localized in a single domain of the protein. We developed a flow cytometry-based assay to compare cell membrane expression of obesity-associated MC4R mutants. Using this assay, we demonstrate that over 54% of the obesity-associated MC4R mutations impair the membrane expression of MC4R. All other mutations impair the basal constitutive activity and/or the EC(50) for the physiological agonist alpha-MSH as measured in a cAMP- dependent luciferase assay. The extent of the alterations in receptor activity ranges from a total suppression of MC4R activation in response to alpha-MSH to a mild alteration of the basal constitutive activity of the receptor. Since most patients are heterozygous for MC4R mutations, these data indicate that a small decrease in overall MC4R activity can cause obesity, strongly supporting the hypothesis that the MC4R is a critical component of the adipostat in humans.

G-protein-coupled receptors (GPCRs) are the largest known family of cell surface receptors, and they control many important physiological events, including sensory perception, chemotaxis, neurotransmission, and energy homeostasis. However, GPCR signaling can be difficult to study in vivo because of the multitude of GPCRs, the lack of specific synthetic agonists, and the fact that some GPCRs activate multiple signaling pathways. One method to circumvent these problems is to develop an engineered receptor that is unresponsive to its endogenous agonist, yet can be fully activated by synthetic, small-molecule drugs. Such a receptor, called a receptor activated solely by a synthetic ligand (RASSL), can be rapidly and reversibly activated by a small-molecule drug and would be a powerful tool to control G-protein signaling in vivo. Here we present the development of a G(s)-coupled RASSL based on the melanocortin-4 receptor (MC4R). MC4R couples exclusively to G(s) at physiologically relevant concentrations of its endogenous ligand, alpha-melanocyte-stimulating hormone (alpha-MSH). Data from human patients and structure-activity studies have shown that several mutations in MC4R cause a decreased affinity for alpha-MSH and can be exploited for RASSL development. Synthetic, small-molecule agonists of MC4R are now available and can be used to activate mutated receptors in vivo. We are engineering a series of mutations in MC4R to remove the peptide-binding site while retaining small-molecule binding and activation. The MC4R G(s) RASSL could be used to control many physiological responses associated with G(s) signaling such as heart rate, energy homeostasis, and cell proliferation.

Progressive renal interstitial fibrosis and tubular atrophy represent the final injury pathway for all commonly encountered forms of renal disease that lead to end-stage renal failure. It has been recently recognized that myofibroblastic cells are the major contributors to the deposition of interstitial collagens. While there are several potential cellular sources of myofibroblasts, attention has focused on the transformation of the organized tubular epithelium to the myofibroblastic phenotype, a process potently driven both in vitro and in vivo by transforming growth factor-beta1 (TGF-beta1). Integrity of the underlying basal lamina provides cellular signals that maintain the epithelial phenotype, and disruption by discrete proteases could potentially initiate the transformation process. We demonstrate that TGF-beta1 coordinately stimulates the synthesis of a specific matrix metalloproteinase, gelatinase A (MMP-2), and its activator protease, MT1-MMP (MMP-14), and that active gelatinase A is absolutely required for epithelial-mesenchymal transformation induced by TGF-beta1. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in the absence of exogenous TGF-beta1. Gelatinase A may also mediate epithelial-mesenchymal transformation in a paracrine manner through the proteolytic generation of active TGF-beta1 peptide. MT1-MMP and gelatinase A were co-localized to sites of active epithelial-mesenchymal transformation and basal lamina disruption in the rat remnant kidney model of progressive renal fibrosis. These studies indicate that a discrete matrix metalloproteinase, gelatinase A, is capable of inducing the complex genetic rearrangements that characterize renal tubular epithelial-mesenchymal transformation.

BACKGROUND

Asthma is a common and costly health condition, but most estimates of its economic effect have relied on secondary sources with limited condition-specific detail.

OBJECTIVE

We sought to estimate the magnitude of direct and indirect costs of adult asthma from the perspective of society.

METHODS

We used cross-sectional survey data from an ongoing community-based panel study of 401 adults with asthma originally derived from random samples of northern California pulmonologists, allergist-immunologists, and family practitioners to assess health care use for asthma, to assess purchase of items to assist with asthma care, and to measure work and other productivity losses. Unit costs derived from public-use and proprietary data sources were then assigned to the survey items.

RESULTS

Total per-person annual costs of asthma averaged $4912 US dollars, with direct and indirect costs accounting for $3180 US dollars (65%) and $1732 US dollars (35%), respectively. The largest components within direct costs were pharmaceuticals ($1605 US dollars [50%]), hospital admissions ($463 US dollars[15%]), and non-emergency department ambulatory visits ($342 US dollars [11%]). Within indirect costs, total cessation of work accounted for $1062 US dollars (61%), and the loss of entire work days among those remaining employed accounted for another $486 US dollars (28%). Total per-person costs were $2646, $4530, and $12,813 US dollars for persons self-reporting mild, moderate, and severe asthma, respectively (P <.0001, 1-way ANOVA).

CONCLUSION

Asthma-related costs are substantial and are driven largely by pharmaceuticals and work loss.

STUDY OBJECTIVES

Laboratory-based spirometry is the "gold standard" for the assessment of lung function, both in clinical and research protocols. These spirometers, however, are neither practical nor affordable for home-based monitoring or studies that collect data in multiple locations. Traditionally, peak flowmeters have been used, but they have important limitations.

DESIGN

Based on data from a cohort of 92 children with asthma, we evaluated the agreement between a portable spirometer and a office-based spirometer, using an in-line technique to evaluate measures from the same effort. We compared a range of pulmonary function parameters collected during office-based tests, and also evaluated whether adequate adherence and data quality could be achieved in a home-based study of children with asthma.

RESULTS

The agreement between the devices for the actual values of peak expiratory flow, FEV(1), and forced expiratory flow at 25% of FVC was excellent. The portable device was programmed with customized software to grade each curve using revised American Thoracic Society acceptability and reproducibility criteria. For 74% of the curves, quality grade agreed with a grade assigned by physician review of the curve from the office-based spirometer. During 2 weeks of twice-daily monitoring at home, children completed an average of 23 of 28 possible sessions (83%). Of these, 84% had at least two acceptable and two reproducible curves. Although children >or= 8 years old were not more adherent, they were significantly more likely to achieve acceptable and reproducible curves.

CONCLUSIONS

Portable spirometers can provide measurements that are highly comparable to those obtained from "gold standard" laboratory spirometers, and high-quality tracings can be achieved both at home and in the office setting. Visual inspection of the curves by experienced reviewers identified unacceptable curves that were not rejected by the quality control software. Portable spirometers are an important contribution to epidemiologic and clinical studies that require frequent measures of a more broad range of pulmonary function parameters than can be provided by peak flowmeters.

In male rats, activity in subdiaphragmatic vagal afferents modulates nociception via an adrenal medulla-dependent mechanism. Because both the vagus and adrenal medullae are sexually dimorphic, we evaluated vagotomy-induced changes in mechanical nociceptive threshold and inflammatory hyperalgesia in female rats and compared them to those previously reported in male rats. We have found that (1) mechanical nociceptive threshold is lower in female rats than in male rats, perhaps because of tonic release of adrenal medullary factors in female rats; (2) mechanical nociceptive threshold in female rats is influenced to a lesser degree by activity in the subdiaphragmatic vagus; (3) vagotomy-induced enhancement of bradykinin hyperalgesia is greater in female rats; (4) in female rats, in contrast to male rats, celiac plus celiac accessory branch vagotomy failed to fully account for the enhancement of bradykinin hyperalgesia in complete subdiaphragmatic vagotomy; and (5) in female rats, in contrast to male rats, adrenal medullectomy plus subdiaphragmatic vagotomy only partially (approximately 30%) reversed the effect of vagotomy on bradykinin hyperalgesia. These findings demonstrate sexual dimorphism in the modulation of both mechanical nociceptive threshold and bradykinin-induced hyperalgesia by activity in subdiaphragmatic vagal afferents as well as the adrenal medulla.

In this study, we investigated the possible involvement of acyl-CoA, reactive intermediary metabolites of 2-arylpropionic acids (profens), in protein adduct formation in rat liver homogenate and in human serum albumin (HSA) in buffer. (RS)-[1-14C]-2-Phenylpropionic acid (14C-2-PPA, 1 mM) was incubated with rat liver homogenate (1.5 mg/ml) in the presence of cofactors of acyl-CoA formation (Mg2+, ATP, and CoA). Aliquots of the incubation mixture were analyzed for covalent binding and acyl-CoA formation over a 3-h period. High-performance liquid chromatographic analysis of the products from such incubations showed the presence of 2-phenylpropionyl-S-acyl-CoA (2-PPA-CoA), which was confirmed by coelution with authentic 2-PPA-CoA, as well as by mass spectrometry. In the same incubations, 2-PPA was shown to bind covalently to hepatic proteins in a time- and ATP-dependent fashion. Inhibition of 2-PPA-CoA formation by acyl-CoA synthetase inhibitors, such as palmitic acid, lauric acid, octanoic acid, and ibuprofen, markedly decreased the extent of covalent binding of 2-PPA to hepatic proteins. Results from these in vitro studies strongly suggest that acyl-CoA thioester derivatives are chemically reactive and are able to bind covalently to tissue proteins in vitro, and, therefore, may contribute significantly to covalent adduct formation of profen drugs in vivo.