HLA-DRB1*04 gene polymorphisms and expressions profiles of interleukin-18 and interleukin-18 binding protein following in vitro stimulation in human peripheral blood mononuclear cells of healthy individuals and patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic arthritic condition that can lead to deformities and disabilities. Interleukin-18 (IL-18) is a proinflammatory cytokine known to play a role in the acute and chronic inflammatory phases of RA. IL-18 binding protein is the natural antagonist of IL-18 protein. We aim to identify the effect of HLA-DRB1*04 gene polymorphisms on IL-18 and IL-18BP gene expressions profiles as well as the time-course profiles following in vitro stimulation with mitogens. Peripheral blood mononuclear cells from 16 RA patients and 21 healthy controls were cultured for 1, 4, 8, 12, 24, 48 and 72 h following stimulation with either LPS or PHA. mRNA expression of IL-18 and IL 18BP were determined by quantitative real-time PCR using a comparative Ct (threshold cycle) method. IL-18 levels in supernatants were measured by enzyme-linked immunosorbent assay. Basal mRNA (4.5-fold) and protein levels of IL-18 were increased and IL-18BP mRNA expression was decreased (8-fold) in RA patients when compared to controls. Similarly, increased IL-18 levels were observed in active RA patients, whereas IL-18BP expression was increased in inactive patients. There was an increase in mRNA and protein levels of IL-18 in RA patients that peaked at 4 h and 8 h respectively following LPS stimulation. A similar profile was observed for IL-18BP; however, the expression level was higher in controls than RA patients. Persistent high production of IL-18 in RA is associated with disease progression and IL-18 BP seems to inhibit this activity.     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

Anionic lipids enriched at the ExPortal of Streptococcus pyogenes

The ExPortal of Streptococcus pyogenes is a membrane microdomain dedicated to the secretion and folding of proteins. We investigated the lipid composition of the ExPortal by examining the distribution of anionic membrane phospholipids. Staining with 10-N-nonyl-acridine orange revealed a single microdomain enriched with an anionic phospholipid whose staining characteristics and behavior in a cardiolipin-deficient mutant were characteristic of phosphatidylglycerol. Furthermore, the location of the microdomain corresponded to the site of active protein secretion at the ExPortal. These results indicate that the ExPortal is an asymmetric lipid microdomain, whose enriched content of anionic phospholipids may play an important role in ExPortal organization and protein trafficking.     

GPIHBP1: an endothelial cell molecule important for the lipolytic processing of chylomicrons

PURPOSE OF REVIEW: To summarize recent data indicating that glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) plays a key role in the lipolytic processing of chylomicrons. RECENT FINDINGS: Lipoprotein lipase hydrolyses triglycerides in chylomicrons at the luminal surface of the capillaries in heart, adipose tissue, and skeletal muscle. The endothelial cell molecule that facilitates the lipolytic processing of chylomicrons has never been clearly defined. Mice lacking GPIHBP1 manifest chylomicronemia, with plasma triglyceride levels as high as 5000 mg/dl. In wild-type mice, GPIHBP1 is expressed on the luminal surface of capillaries in heart, adipose tissue, and skeletal muscle. Cells transfected with GPIHBP1 bind both chylomicrons and lipoprotein lipase avidly. SUMMARY: The chylomicronemia in Gpihbp1-deficient mice, the fact that GPIHBP1 is located within the lumen of capillaries, and the fact that GPIHBP1 binds lipoprotein lipase and chylomicrons suggest that GPIHBP1 is a key platform for the lipolytic processing of triglyceride-rich lipoproteins.     

Identification of proteins involved in microglial endocytosis of alpha-synuclein

Aggregated alpha-synuclein, a protein playing pivotal roles in the pathogenesis of Parkinson disease (PD) and related synucleinopathy, has been shown to activate microglia, the key cells in neuroinflammation. However, the mechanisms by which aggregated alpha-synuclein enters microglia remain uncharacterized. In this study, we first replicated our previous results with a modified protocol that generated aggregated alpha-synuclein more efficiently. Next, using two recently developed proteomic techniques, SILAC (Stable Isotope Labeling of Amino acid in Cell cultures) and PROCEED (PROteome of Cell Exposed Extracellular Domains), we studied the plasma membrane proteins of primary cultured microglia that might be interacting with aggregated alpha-synuclein and mediating its internalization. The results demonstrated that 250 nM alpha-synuclein, aged for 6 h with a magnetic stir bar, was just as potent in activating microglia as the aggregated alpha-synuclein produced by aging without constant agitation for 7 days. The proteomic analysis identified 111 membrane proteins; of these, 46 proteins were altered in relative abundance in the membrane compartment after treatment with aggregated alpha-synuclein for 3 h. Two of these proteins, clathrin and calnexin, were further evaluated with Western blotting, demonstrating good agreement with quantitative proteomics. Finally, immunocytochemical as well as co-immunoprecipitation studies indicated that clathrin was indeed co-localized with internalized alpha-synuclein in microglia. These results suggest for the first time that microglial activation secondary to internalization of aggregated alpha-synuclein likely requires participation of clathrin, which is an essential protein of the polyhedral coat of coated pits and vesicles that play major roles in endocytosis and vesicular trafficking.     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.