Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Identification and functional analysis of novel human melanocortin-4 receptor variants.

Inactivation of the melanocortin-4 receptor (MC4-R) by gene-targeting results in mice that develop maturity-onset obesity, hyperinsulinemia, and hyperglycemia. These phenotypes resemble common forms of human obesity, which are late-onset and frequently accompanied by NIDDM. It is not clear whether sequence variation of the MC4-R gene contributes to obesity in humans. Therefore, we examined the human MC4-R gene polymorphism in 190 individuals ascertained on obesity status. Three allelic variants were identified, including two novel ones, Thr112Met and Ile137Thr. To analyze possible functional alterations, the variants were cloned and expressed in vitro and compared with the wild-type receptor. One of the novel variants, Ile137Thr, identified in an extremely obese proband (BMI 57), was found to be severely impaired in ligand binding and signaling, raising the possibility that it may contribute to development of obesity. Furthermore, our results also suggest that sequence polymorphism in the MC4-R coding region is unlikely to be a common cause of obesity in the population studied, given the low frequency of functionally significant mutations.

Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story.

A plasmid-borne transposon encodes enzymes and regulator proteins that confer resistance of enterococcal bacteria to the antibiotic vancomycin. Purification and characterization of individual proteins encoded by this operon has helped to elucidate the molecular basis of vancomycin resistance. This new understanding provides opportunities for intervention to reverse resistance.

Kinetically Selective Inhibitors of Histone Deacetylase 2 (HDAC2) as Cognition Enhancers.

Aiming towards the development of novel nootropic therapeutics to address the cognitive impairment common to a range of brain disorders, we set out to develop highly selective small molecule inhibitors of HDAC2, a chromatin modifying histone deacetylase implicated in memory formation and synaptic plasticity. Novel ortho-aminoanilide inhibitors were designed and evaluated for their ability to selectively inhibit HDAC2 versus the other Class I HDACs. Kinetic and thermodynamic binding properties were essential elements of our design strategy and two novel classes of ortho-aminoanilides, that exhibit kinetic selectivity (biased residence time) for HDAC2 versus the highly homologous isoform HDAC1, were identified. These kinetically selective HDAC2 inhibitors (BRD6688 and BRD4884) increased H4K12 and H3K9 histone acetylation in primary mouse neuronal cell culture assays, in the hippocampus of CK-p25 mice, a model of neurodegenerative disease, and rescued the associated memory deficits of these mice in a cognition behavioural model. These studies demonstrate for the first time that selective pharmacological inhibition of HDAC2 is feasible and that inhibition of the catalytic activity of this enzyme may serve as a therapeutic approach towards enhancing the learning and memory processes that are affected in many neurological and psychiatric disorders.

Melanocortin and leptin signaling systems: central regulation of catabolic energy balance.

The recent cloning of the ob gene (leptin) has revolutionized our understanding of obesity and the underlying factors that govern weight homeostasis. There is growing evidence that long term food intake regulation is controlled by the central nervous system by a number of peptide hormones in response to changes in leptin levels. Studies of these hormones, using both genetic and pharmacological approaches, have provided a foundation for decoding the molecular logic of the neuronal circuits which regulate food intake control and energy balance. A review of the current progress in the melanocortin-4 receptor pathway, with particular emphasis on its relation to leptin, neuropeptide Y and other obesity hormones known to modulate weight homeostasis, is presented.

Melanocortin-4 receptor: a novel signalling pathway involved in body weight regulation.

For many years, genetically obese mouse strains have provided models for human obesity. The Avy/-agouti mouse, one of the oldest obese mouse models, is characterized by maturity-onset obesity and diabetes as a result of ectopic expression of the secreted protein hormone, agouti protein. Agouti protein is normally expressed in hair follicles to regulate pigmentation through antagonism of the melanocortin-1 receptor, but in-vitro studies have demonstrated that the hormone also has potent antagonist activity for the melanocortin-4 receptor (MC4-R). Subsequent development of the MC4-R knockout mouse model demonstrated that MC4-R plays a role in weight homeostasis as these mice recapitulated the metabolic defects of the agouti mouse. Further evidence for this hypothesis was obtained from pharmacological studies utilizing peptides with MC4-R agonist activity, that inhibited food intake (when administered intracerebrally). Additional studies with peptide antagonists have now implicated the MC4-R in the leptin signalling pathway. Finally, evidence that the MC4-R may play a role in human obesity has been obtained from the identification of a dis-functional variant of the receptor in genetically obese subjects.

Coordinated home care: the Massachusetts General Hospital experience.

The problem of post-hospital care remains a continued challenge, as many patients who no longer require expensive acute care facilities continue to occupy these beds, awaiting appropriate placement. The Massachusetts General Hospital Coordinated Home Care program, under the central administration of the Boston Visiting Nurse Association, has demonstrated that home care can be a viable, economically feasible alternative to institutionalization for carefully selected patients, when the appropriate medical and social needs can be met. Three major groups of patients have been effectively cared for: 1) patients with multi-system chronic illness; 2) patients with terminal malignancies; and 3) patients with catastrophic neurologic disease. The organization of the Coordinated Home Care program, the criteria for patient selection, and the issue of funding are reviewed. The impact of this program is examined in terms of its potential for better utilization of the Massachusetts General Hospital facilities, as well as the more appropriate coordination and use of existing health care resources in the community.

Immunolocalization of insulin, glucagon, pancreatic polypeptide, and somatostatin in the pancreatic islets of the possum, Trichosurus vulpecula.

Antibodies to insulin, glucagon, pancreatic polypeptide (PP), and somatostatin were used in the immunofluorescence procedure to demonstrate localization of the four hormones in cells of the pancreatic islets of the brushtailed possum, Trichosurus vulpecula. Most pancreatic islets revealed some differences in the topographical distribution and cell number of each endocrine cell type. Insulin immunoreactive cells were observed in most islets where they occurred as groups of cells peripherally and within the islet. In several islets glucagon cells were the predominant cell population and were distributed peripherally as well as centrally. Pancreatic polypeptide cells were fewer in number and usually occurred as single cells within the islet. Cells immunoreactive to antisomatostatin serum were observed in varying numbers in the peripheral and central regions of the islet. The present immunofluorescence study demonstrates differences in the topographical distribution of the four major pancreatic hormones between a marsupial species and several of the eutherian mammals.

Longitudinal study of first phase insulin release in the BB rat.

First phase insulin release was measured in response to intravenous glucose given weekly from approximately day 40 in 6 BB rats which subsequently developed diabetes and in age-matched non-diabetic (n = 15) and normal Wistar rats (n = 8) until day 180. The mean sequential insulin responses in BB rats with and without diabetes were significantly lower (p = 0.008 and less than 0.0001, respectively) than in normal rats from an early age. Five diabetic BB rats showed a progressive decline in first phase insulin release immediately prior to glycosuria, with the impaired phases ranging from 25-50 days. However, protracted periods of low first phase responses were also seen in several aglycosuric BB rats, which showed histological evidence of insulitis and B-cell loss. Our findings demonstrate that, although most BB rats with diabetes show a progressive impairment of B-cell function preceding the disease, this aberrant phase can also be present in BB rats which remain aglycosuric. Impaired first phase insulin release in response to serial intravenous glucose tolerance tests may not be a reliable predictor of Type 1 (insulin-dependent) diabetes in this animal model.

Cross-talk between the histidine protein kinase VanS and the response regulator PhoB. Characterization and identification of a VanS domain that inhibits activation of PhoB.

VanS is a two-component transmembrane sensory kinase that, together with its response regulator VanR, activates the expression of genes responsible for vancomycin resistance in Enterococcus faecium BM4147. In this report, we demonstrate that the cytoplasmic domain of VanS (including residues Met95 to Ser384) is capable of high level activation (> 500 fold) of the Escherichia coli response regulator PhoB in vivo in the absence of its signaling kinases PhoR, CreC (PhoM), or acetyl phosphate synthesis. In vitro experiments carried out on the purified proteins confirmed that the activation is due to efficient cross-talk between VanS and PhoB, since phospho-VanS catalyzed transfer of its phosphoryl group to PhoB with approximately 90% transfer in 5 min at a 1:4 VanS/PhoB stoichiometry. However, the rate of transfer was at least 100-fold slower than that observed between phospho-VanS and VanR. The in vivo activation of PhoB was used as a reporter system to identify peptide fragments of VanS capable of interfering with activation by VanS(Met95-Ser384), in order to identify an interaction domain. A library of plasmids encoding fragments of VanS(Met95-Ser384) was constructed using transposon mutagenesis, and a subpopulation of these plasmids encoded peptides that interfered with activation of PhoB by VanS(Met95-Ser384). A minimal size fragment (Met95-Ile174) was shown to be both necessary and sufficient for potent inhibition (85%) of this activation.

Biochemical characterization of a phosphinate inhibitor of Escherichia coli MurC.

The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.

Kinetic comparison of the specificity of the vancomycin resistance VanSfor two response regulators, VanR and PhoB.

Induction of vancomycin resistance in the Gram-positive Enterococci requires a two-componet regulatory system, VanS and VanR, for transcriptional activation of three genes (vanH, A, X) that encode enzymes for a cell wall biosynthetic pathway that produces an altered peptidoglycan intermediate with lower affinity for the antibiotic. The catalytic efficiency (kcat/KM) has been determined for phosphotransfer from the phosphohistidyl form of VanS to both its homologous partner VanR and the heterologous (Escherichia coli) response regulator Phob. The rate of formation of the phosphoaapartyl forms of VanR and PhoB were determined as well as the rate of appearance of inorganic phosphate. Using PhoB in excess of P-VanS, a pseudo-first-order rate constant (kxfer) of 0.2 min-1 for phosphotransfer and a KM for PhoB of 100 microM were readily determined. The corresponding kxfer of 96 min-1 for phosphotransfer from P-VanS to VanR required quench kinetics. A KM of 3 microM was estimated for VanR, leading to a 10(4)-fold preference in kxfer/KM for phosphotransfer to VanR compared to PhoB. No phosphotransfer was detachable to three other E. coli response regulators, OmpR, ArcB, or CreB, providing some sense of the selectivity against two-component regulatory system cross-talk. In the phosphotransfer from P-VanS to PhoB and VanR, there was evidence of competition between water, to give Pi, and the specific aspartyl beta-COO- moiety of either PhoB or VanR with about 25% of the initial flux generating inorganic phosphate. The kinetics of phosphotransfer from P-VanS to VanR were complicated by inhibition by free VanS but, the inhibition pattern could be modeled to yield at KD of 30 nM for VanR binding to free VanS, an affinity similar to that of the CheA-CheY pair in E. coli chemotaxis.

Transcriptional regulation of the Enterococcus faecium BM4147 vancomycin resistance gene cluster by the VanS-VanR two-component regulatory system in Escherichia coli K-12.

An Escherichia coli K-12 model system was developed for studying the VanS-VanR two-component regulatory system required for high-level inducible vancomycin resistance in Enterococcus faecium BM4147. Our model system is based on the use of reporter strains with lacZ transcriptional and translational fusions to the PvanR or PvanH promoter of the vanRSHAX gene cluster. These strains also express vanR and vanS behind the native PvanR promoter, the arabinose-inducible ParaB promoter, or the rhamnose-inducible PrhaB promoter. Our reporter strains have the respective fusions stably recombined onto the chromosome in single copy, thereby avoiding aberrant regulatory effects that may occur with plasmid-bearing strains. They were constructed by using allele replacement methods or a conditionally replicative attP plasmid. Using these reporter strains, we demonstrated that (i) the response regulator VanR activates PvanH, but not PvanR, expression upon activation (phosphorylation) by the partner kinase VanS, the noncognate kinase PhoR, or acetyl phosphate, indicating that phospho-VanR (P-VanR) is a transcriptional activator; (ii) VanS interferes with activation of VanR by PhoR or acetyl phosphate, indicating that VanS also acts as a P-VanR phosphatase; and (iii) the conserved, phosphate-accepting histidine (H164) of VanS is required for activation (phosphorylation) of VanR but not for deactivation (dephosphorylation) of P-VanR. Similar reporter strains may be useful in new studies on these and other interactions of the VanS-VanR system (and other systems), screening for inhibitors of these interactions, and deciphering the molecular logic of the signal(s) responsible for activation of the VanS-VanR system in vivo. Advantages of using an E. coli model system for in vivo studies on VanS and VanR are also discussed.

Altered recognition mutants of the response regulator PhoB: a new genetic strategy for studying protein-protein interactions.

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a "patch" near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and P1 signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6K gamma attP "genome targeting" suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Central infusion of melanocortin agonist MTII in rats: assessment of c-Fos expression and taste aversion.

Like leptin (OB protein), central infusion of the nonspecific melanocortin agonist MTII reduces food intake for relatively long periods of time (i.e., 12 h; W. Fan, B. A. Boston, R. A. Kesterson, V. J. Hruby, and R. D. Cone, Nature; 385: 165-168, 1997). To test the hypothesis that MTII may influence ingestive behavior via mechanisms similar to those that mediate the effects of leptin, we infused a single dose of MTII into the third ventricle (i3vt) of Long-Evans rats and examined three dependent measures that have been studied following i3vt infusion of leptin: 1) effects on long-term food intake and body weight (48 h), 2) patterns of c-Fos expression in the brain, and 3) conditioned taste aversion learning. Similar to leptin, MTII reduced 48-h food intake (1.0 nmol dose), reduced body weight at 24 and 48 h (0.1 and 1.0 nmol doses, respectively), and induced c-Fos expression in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala. In contrast to leptin, MTII was found to produce conditioned taste aversions. These results are consistent with the hypothesis that MTII may influence regulatory behavior via mechanisms similar to those that mediate the effects of leptin.

The N-terminal domain of RGS4 confers receptor-selective inhibition of G protein signaling.

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi alpha subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10(4)-fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Galphaq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling.