Discovery and structure-activity relationships of urea derivatives as potent and novel CCR3 antagonists

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist.     

The insulin/PI 3-kinase pathway regulates salt chemotaxis learning in Caenorhabditis elegans

The insulin-like signaling pathway is known to regulate fat metabolism, dauer formation, and longevity in Caenorhabditis elegans. Here, we report that this pathway is also involved in salt chemotaxis learning, in which animals previously exposed to a chemoattractive salt under starvation conditions start to show salt avoidance behavior. Mutants of ins-1, daf-2, age-1, pdk-1, and akt-1, which encode the homologs of insulin, insulin/IGF-I receptor, PI 3-kinase, phosphoinositide-dependent kinase, and Akt/PKB, respectively, show severe defects in salt chemotaxis learning. daf-2 and age-1 act in the ASER salt-sensing neuron, and the activity level of the DAF-2/AGE-1 pathway in this neuron determines the extent and orientation of salt chemotaxis. On the other hand, ins-1 acts in AIA interneurons, which receive direct synaptic inputs from sensory neurons and also send synaptic outputs to ASER. These results suggest that INS-1 secreted from AIA interneurons provides feedback to ASER to generate plasticity of chemotaxis.     

Genetic Evidence Linking Age-Dependent Attenuation of the 26S Proteasome with the Aging Process

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies'' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.Ubiquitin-conjugated, misfolded protein aggregates are observed in the brain during normal aging and in late-onset human neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, and polyglutamine diseases (e.g., Huntington''s disease or spinocerebellar ataxias) (9). Many of the mutations that cause dominantly inherited neurodegenerative diseases dramatically increase the amount of protein aggregates in vitro and in vivo, supporting the widely accepted hypothesis that proteotoxicity caused by the aggregates underlies the pathogenesis of many neurodegenerative diseases (32). Proteotoxicity can have many effects, including disruption of microtubule-dependent axonal transport (10), perturbation of membrane permeability (23), and impaired function of the ubiquitin-proteasome system (UPS) (1, 17). Aggregation-mediated toxicity has also been suggested in normal aging, because recent reports show that the impairment of autophagy in the central nervous system causes accumulation of ubiquitinated proteins and leads to neurodegenerative diseases (12, 21). These observations suggest that the continuous clearance of misfolded proteins through cellular degradation systems, including the UPS and autophagy, is important for preventing aggregation-mediated proteotoxicity both in age-related neurodegenerative diseases and in normal aging.Clinical symptoms of neurodegenerative diseases generally do not appear or progress until advanced ages, not only in sporadic forms but also in inherited forms of neurodegenerative diseases (26). These observations suggest that aggregation-mediated toxicity appears in a late-onset manner both in normal aging and in neurodegenerative diseases. Furthermore, a link between the aging process and aggregation-mediated proteotoxicity has been suggested by evidence that Huntington''s disease-associated proteotoxicity was ameliorated when the aging process slowed, that is, the life span extension via decreased insulin/insulin growth factor-1-like signaling in Caenorhabditis elegans (13, 31).A possible mechanism for the late onset of aggregation-mediated toxicity is age-related impairment of the UPS, because loss-of-function mutations in genes encoding UPS components can enhance the cytotoxicity of protein aggregation in dominantly inherited neurodegenerative diseases (4, 5, 18). In addition, an age-related decline of proteasome activity has been observed in the tissues of humans and other mammals (8) and in aged flies (36). Considering the role of the proteasome in neuroprotection and the age dependence of most neurodegenerative diseases, the age-related decline of proteasome activity could well be a key factor both in normal aging and in the late onset and/or progression of neurodegenerative diseases. However, the mechanism underlying the age-related decline of proteasome activity remains to be elucidated, and there is no direct genetic evidence showing that the age-related decline of proteasome activity causes age-related aggregation-mediated toxicity in normal aging and in age-related neurodegenerative diseases.Here, we studied the age-related decline of proteasome activity by using Drosophila melanogaster and found age-related attenuation of the 26S proteasome activity and abundance that was associated with impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we identified Rpn11, which encodes one of the lid subunits in the 19S RP, as a suppressor of the age-dependent progression of a polyglutamine-induced neurodegenerative phenotype. The overexpression of Rpn11 prevented the age-related reduction of the 26S proteasome activity, which suppressed the age-dependent accumulation of ubiquitinated proteins and extended the life span. On the other hand, the loss of function of Rpn11 enhanced the age-related reduction of 26S proteasome activity, leading to a shorter life span, a premature age-dependent accumulation of ubiquitinated proteins, and an early onset of a neurodegenerative phenotype. Our results demonstrate for the first time that the age-related reduction of the 26S proteasome activity is a key factor in the induction of certain age-related biological changes and in the increased risk for the onset or progression of neurodegenerative diseases. Our findings imply that improving the amount and/or activity of the 26S proteasome by overexpressing a lid subunit, such as Rpn11, could provide an extension to the mean life span and prevent the age-dependent onset or progression of neurodegeneration.     

Neuroblastoma GOTO cells are hypersensitive to disruption of lipid rafts

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-β-cyclodextrin (MβCD); the GM2-rich rafts and sensitivity to MβCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MβCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells. Until this study, GM2-rich rafts are poorly characterized and MβCD hypersensitivity, which may have clinical implications, has not been reported.     

Reversal of Salt Preference Is Directed by the Insulin/PI3K and Gq/PKC Signaling in Caenorhabditis elegans

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the G_q signaling pathway composed of G_qα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.ANIMALS show various behaviors in response to environmental cues and modulate behaviors according to previous experience. To understand neuronal plasticity underlying learning, it is important to dissect neurons and molecules for sensing environmental stimuli, storing memory, and executing learned behaviors.The nematode Caenorhabditis elegans has only 302 neurons and functions of sensory neurons are well characterized (White et al. 1986; Bargmann 2006). C. elegans is attracted to odorants sensed by the AWC olfactory neurons or to salts sensed by the ASE gustatory neurons (Bargmann and Horvitz 1991; Bargmann et al. 1993). The ASE neuron class consists of a bilaterally symmetrical pair, ASE-left (ASEL) and ASE-right (ASER), which sense different sets of ions including Na⁺ and Cl⁻, respectively (Pierce-Shimomura et al. 2001; Suzuki et al. 2008; Ortiz et al. 2009). ASEL is activated by an increase in salt concentration, whereas ASER is activated by a decrease in salt concentration (Suzuki et al. 2008). In the ASE gustatory neurons, a cyclic GMP (cGMP) signaling pathway mediates sensory transduction (Komatsu et al. 1996; Suzuki et al. 2008; Ortiz et al. 2009). ASEL and ASER express different sets of receptor-type guanylyl cyclases (gcys) (Ortiz et al. 2006). Of these, gcy-22, which is specifically expressed in ASER, is important for attraction to ASER-sensed ions such as Cl⁻ (Ortiz et al. 2009).Preference for salts changes according to previous experience (known as gustatory plasticity or salt chemotaxis learning) (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). When worms are grown on a medium that contains sodium chloride (NaCl) and food (Escherichia coli), they show attraction to NaCl by using ASE neurons (Bargmann and Horvitz 1991; Suzuki et al. 2008). In contrast, after exposure to the salt under starvation conditions, they show reduced attraction to or even avoid the salt (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). In C. elegans, it was proposed that preference for a sensory cue is defined by the sensory neuron that detects the cue (Troemel et al. 1997). ASE neurons play a major role for salt attraction (Bargmann and Horvitz 1991; Suzuki et al. 2008; Ortiz et al. 2009). However, little is known about sensory neurons that drive the learned salt avoidance; it remains unclear whether ASE neurons act as salt receptors for the learned avoidance.We have previously shown that an insulin/PI 3-kinase signaling pathway is essential for salt chemotaxis learning (Tomioka et al. 2006). In C. elegans, the insulin-like signaling is composed of daf-2, age-1, and akt-1, which encode homologs of insulin receptor, PI 3-kinase, and protein kinase B, respectively (Morris et al. 1996; Kimura et al. 1997; Paradis and Ruvkun 1998). Mutants of daf-2, age-1, and akt-1 show attraction to salt even after starvation/NaCl conditioning (Tomioka et al. 2006).daf-18 encodes a homolog of phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate and counteracts the insulin/PI 3-kinase signaling (Ogg and Ruvkun 1998; Gil et al. 1999; Mihaylova et al. 1999; Rouault et al. 1999; Solari et al. 2005). Mutants of daf-18, in which the PI 3-kinase signaling is activated, show reduced attraction to NaCl even without conditioning. Since the insulin/PI 3-kinase signaling acts in ASER, we proposed that the insulin/PI 3-kinase signaling attenuates the attractive drive of ASER (Tomioka et al. 2006).In C. elegans, diacylglycerol (DAG) regulates functions of motor neurons and sensory neurons. egl-30, which encodes the α-subunit of heterotrimeric G-protein G_q, facilitates production of DAG and enhances locomotory movements (Brundage et al. 1996; Lackner et al. 1999). In the AWC olfactory neurons, a novel protein kinase C-ɛ/η (nPKC-ɛ/η) ortholog TTX-4 (also known as PKC-1), which is one of DAG targets, plays an essential role in attraction behavior to AWC-sensed odors (Okochi et al. 2005; Tsunozaki et al. 2008). GOA-1 G_oα regulates olfactory adaptation by antagonizing G_qα–DAG signaling (Matsuki et al. 2006).This study investigated the involvement of the ASE taste receptor neurons in the starvation-induced salt avoidance. We show that both ASEL and ASER contribute to salt chemotaxis learning. Activation of the PI 3-kinase signaling and the G_q/DAG/PKC signaling acted antagonistically in reversal of ASER function, whereas these signaling pathways did not have prominent effects on ASEL function. In ASER, GCY-22 was required for both salt attraction and avoidance. These results suggest that ASE neurons are important for bidirectional chemotaxis and also suggest that distinct molecular mechanisms regulate functions of ASEL and ASER in salt chemotaxis learning.     

Synthesis and evaluation of opioid receptor-binding affinity of elaeocarpenine and its analogs

Both enantiomers of elaeocarpenine (1) and its analogs, 21, 22, 25, and 27, were synthesized from bicyclic aldehydes 8–10 via a flexible route previously established for total synthesis of grandisines, and their binding affinities for μ-, κ- and δ-opioid receptor subtypes were evaluated. We found that (9R)-1 exhibited higher affinity than (9S)-1 for all the subtypes, but the enantiomers showed little subtype selectivity. Analogs 21 having a pyrrolizidine skeleton and 27 having a stemona-type skeleton in place of the indolizidine unit of (9S)-1 showed μ-selective and μ-, κ-selective binding, respectively.     

Galanin-like peptide and the regulation of feeding behavior and energy metabolism

The hypothalamic neuropeptides modulate physiological activity via G protein-coupled receptors (GPCRs). Galanin-like peptide (GALP) is a 60 amino acid neuropeptide that was originally isolated from porcine hypothalamus using a binding assay for galanin receptors, which belong to the GPCR family. GALP is mainly produced in neurons in the hypothalamic arcuate nucleus. GALP-containing neurons form neuronal networks with several other types of peptide-containing neurons and then regulate feeding behavior and energy metabolism. In rats, the central injection of GALP produces a dichotomous action that involves transient hyperphasia followed by hypophasia and a reduction in body weight, whereas, in mice, it has only one action that reduces both food intake and body weight. In the present minireview, we discuss current evidence regarding the function of GALP, particularly in relation to feeding and energy metabolism. We also examine the effects of GALP activity on food intake, body weight and locomotor activity after intranasal infusion, a clinically viable mode of delivery. We conclude that GALP may be of therapeutic value for obesity and life-style-related diseases in the near future.     

Induction and enhancement of stress proteins in a trichloroethylene-degrading methanotrophic bacterium, Methylocystis sp. M

The responses of the trichloroethylene-degrading bacterium Methylocystis sp. M to six different water-pollutants, carbon starvation, and temperature-shock (heat and cold) were examined using 2-dimensional gel electrophoresis. Twenty-eight polypeptides were induced, and these stress-induced proteins were classified into three groups. Some of the chemically induced proteins were the same as those induced by carbon starvation and temperature-shock. Two of the polypeptides were induced by trichloroethylene. Trichloroethylene-stress protein synthesis required 1-2 h at a concentration of trichloroethylene that had no effect on growth. Furthermore, 25 stress-enhanced polypeptides were observed, and one of these was enhanced by trichloroethylene. Based on these results, we discuss applications of chemical-stress induction of proteins to establish effective bioremediation and bioassay by methanotrophs.     

Bax-inhibiting peptide derived from mouse and rat Ku70

Bax is a proapoptotic protein that plays a key role in the induction of apoptosis. Ku70 has activities to repair DNA damage in the nucleus and to suppress apoptosis by inhibiting Bax in the cytosol. We previously designed peptides based on the amino acid sequence of Bax-binding domain of human Ku70, and showed that these peptides bind Bax and inhibit cell death in human cell lines. In the present report, we examined the biological activities of other pentapeptides, VPTLK and VPALR, derived from mouse and rat Ku70. Cells in culture accumulated FITC-labeled VPTLK and VPALR, indicating that these peptides are cell permeable (human, mouse, rat, and porcine cells were examined). These peptides bound to Bax and suppressed cell death in various cell types including primary cultured cells. These data suggest that such Bax inhibiting peptides from three mammalian species may be used to protect healthy cells from apoptotic injury under pathological conditions.