No abstract

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 (1) (hereafter referred to as MCH 1 ) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH 2 , initially identified in a genomic survey sequence as being homologous to MCH 1 receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH 1 and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH 2 receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca 2؉ levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH 2 receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH 1 in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses. Melanin-concentrating hormone (MCH)1 is a cyclic neuropeptide that was first discovered in teleost fish, in which it acts as a skin color-regulating hormone (2). In rodents its tissue distribution in the perikarya of the lateral hypothalamus and the zona incerta suggests that MCH may be involved in a variety of behavioral responses (3). Similar tissue distributions have been reported in both bird (4) and monkey (5). Reports implicating MCH in the regulation of feeding behavior show that increased food intake occurs after direct administration of MCH into the brain (6) and that MCH is up-regulated after fasting and in obese leptin-deficient mice (7). There are also reports that suggest MCH may be involved in aggressive behavior, anxiety, and reproductive function (8, 9).Recently, several groups independently identified a 353-amino acid splice variant of the orphan G protein-coupled receptor (GPCR) SLC-1 (1) as an MCH receptor (10 -14). In view of the findings of the current study, we propose that this form of SLC-1 be hereafter referred to as MCH 1 . Southern blot and related studies have indicated the absence of additional MCH receptor subtypes that closely resemble MCH 1 at the DNA level (3, 10, 15). However, because degeneracy in receptor-ligand pairings throughout the GPCR superfamily is common, we reasoned that other MCH receptors with low homology to the MCH 1 receptor may exist. This suggestion is supported by reports of pharmacological differences between the MCH 1 receptor and MCH binding sites in various cell lines and tissues (10).Sequencing of the human...

A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.

1 The functional pro®le of the long form of the human cloned 5-HT 7 receptor (designated h5-HT 7(a) ) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding pro®le. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT 7(a) receptor. 2 The receptor binding pro®le, determined by competition with [ 3 H]-5-CT, was consistent with that previously reported for the h5-HT 7(a) receptor. The selective 5-HT 7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzene sulfonamide) displayed high anity (pK i 7.5) for the receptor. 3 In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT45-HT48-OH-DPAT) was the same in functional and binding studies. 4 Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA 2 of 7.2+0.2 and slope not signi®cantly dierent from 1, consistent with competitive antagonism. 5 The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding anity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist pro®les compared to the other antagonists tested. These inhibitory eects of antagonists appear to be 5-HT 7 receptor-mediated and to re¯ect inverse agonism. 6 It is concluded that in this expression system, the h5-HT 7(a) receptor shows the expected binding and functional pro®le and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.