Increased circulating levels and salivary gland expression of interleukin-18 in patients with Sjögren's syndrome: relationship with autoantibody production and lymphoid organization of the periductal inflammatory infiltrate

IL-18, an immunoregulatory and proinflammatory cytokine, has been shown to play an important pathogenic role in Th1-driven autoimmune disorders. In this study, we evaluated the circulating levels and salivary-gland expression of IL-18 in patients with Sjögren''s syndrome (SS), a mainly Th1-mediated disease. IL-18 serum levels were measured by ELISA in 37 patients with primary SS, 42 with rheumatoid arthritis, and 21 normal controls. We demonstrated high IL-18 serum levels in SS, similar to those in rheumatoid arthritis patients and significantly higher than in controls (P < 0.01). In addition, IL-18 serum concentrations were significantly higher in anti-SSA/Ro⁺ and anti-SSB/La⁺ than in anti-SSA/Ro^- and anti-SSB/La^- SS patients (respectively, P = 0.01, P < 0.01). Serum IL-18 correlated strongly with anti-SSA/Ro (P = 0.004) and anti-SSB/La (P = 0.01) titers. Salivary gland IL-18 expression was investigated by single/double immunohistochemistry in 13 patients with primary SS and in 10 with chronic sialoadenitis, used as controls. The expression of IL-18 was also examined in periductal inflammatory foci in relation to the acquisition of features of secondary lymphoid organs such as T–B compartmentalization, formation of follicular dendritic cell networks, and presence of germinal-center-like structures. IL-18 expression in SS salivary glands was detected in 28 of 32 periductal foci of mononuclear cells (87.5%), while no IL-18 production by infiltrating cells was detected in patients with chronic sialoadenitis. Within the inflammatory foci, IL-18 immunoreactivity co-localized almost exclusively with CD68⁺ macrophages. In addition, IL-18 was found in 15 of 19 foci (78.9%) with no evidence of T–B cell compartmentalization (nonsegregated) but in 100% of the segregated aggregates, both in T- and B-cell-rich areas. Strikingly, IL-18 was strongly expressed by CD68⁺ tingible body macrophages in germinal-centre-like structures both in SS salivary glands and in normal lymph nodes. IL-18 expression was observed in the ducts of all SS biopsies but in only 4 of 10 patients with nonspecific chronic sialoadenitis (P < 0.01). This study provides the first evidence of increased circulating levels and salivary gland expression of IL-18 in SS, suggesting an important contribution of this cytokine to the modulation of immune inflammatory pathways in this condition.     

Stereospecific cleavage of carbon-phosphorus bonds: stereochemical course of the phosphinoyl curtius (Harger) reaction

The homochiral phosphinic azides (R,R)-1 and (S,S)-1 were prepared in enantiomerically pure form by resolution of diastereomeric phosphinamides derived from (S)-l-phenylethylamine and (R)-phenylglycine. Irradiation of the azides in methanol induced a photo-Curtius rearrangement to phosphonamidates in which the stereogenic carbon unit migrated to a nitrogen atom. Hydrolysis of the phosphonamidates produced 1-phenylethylamine, which was 99.0% e.e. and of the same configuration as the carbon unit in the starting azide (99.0% retention).     

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor.     

Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle

Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.     

Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient’s enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Abstract. Cyclic AMP is known to function as the chemo-tactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyosteliwn discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultra-microtechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the pre-spore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

A Tobacco Etch Virus Protease with Increased Substrate Tolerance at the P1' position

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein–protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1’ Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1’ Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.     

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry,Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.