A role for lymphotoxin in primary Sjogren's disease

The etiology of salivary gland injury in primary Sj?gren's disease is not well understood. We have previously described a mouse model of Sj?gren's disease, IL-14α transgenic (IL14αTG) mice, which reproduces many of the features of the human disease. We now demonstrate a critical role for lymphotoxin α (LTA) in the pathogenesis of Sj?gren's disease in IL14αTG mice. IL14αTG mice express LTA mRNA in their salivary glands and spleen and produce soluble LTA protein in their salivary secretions. When IL14αTG mice were crossed with LTA(-/-) mice, the IL14αTG.LTA(-/-) mice retained normal salivary gland secretions and did not develop either lymphocytic infiltration of their salivary glands or secondary lymphomas. However, both IL14αTG and IL14αTG.LTA(-/-) mice produced similar amounts of IFN-α and had similar deposition of autoantibodies in their salivary glands. Both IL14α and IL14α/LTA(-/-) mice had similar B cell responses to T-dependent and T-independent Ags, L-selectin expression, and expression of RelA, RelB, and NF-κB2 in their spleens. These studies suggest that LTA plays a critical role in the local rather than systemic inflammatory process of Sj?gren's disease. Furthermore, local production of soluble LTA in the salivary glands of IL14αTG mice is necessary for the development of overt Sj?gren's disease. Autoantibody deposition alone is not sufficient to produce salivary gland dysfunction. We also demonstrate that LTA is increased in the salivary gland secretions and sera of patients with Sj?gren's disease, further strengthening the biological relevance of the IL14αTG model to understanding the pathogenesis of human disease.     

Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca²⁺-induced conformational changes of IP₃Rs in their native membrane environment. We found that Ca²⁺ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca²⁺ induces a conformational change in the IP₃R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca²⁺ were observed at ∼0.5 μm and was sensitive to inhibition by IP₃. Ca²⁺ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1–225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca²⁺. However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca²⁺. We conclude the following: a) that large conformational changes induced by Ca²⁺ can be detected in IP₃Rs in situ; b) these changes may be driven by Ca²⁺ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP₃R may be relatively inaccessible to regulatory proteins unless Ca²⁺ is present.     

Pioglitazone prevents cardiac remodeling in high-fat, high-calorie-induced Type 2 diabetes mellitus

The agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARgamma agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARgamma was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi (n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 +/- 0.5 microM in N groups compared with 12.4 +/- 0.6 microM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 +/- 0.02 mm in the D group compared with 0.42 +/- 0.01 mm in the N group. LV diastolic diameter was 3.05 +/- 0.01 mm in the D group compared with 2.20 +/- 0.02 mm in the N group. LV systolic diameter was 1.19 +/- 0.02 mm in the D group and 0.59 +/- 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARgamma activity in high-fat, calorie-induced Type 2 diabetes mellitus.     

The early vertebrate Danio rerio Mr 46000 mannose-6-phosphate receptor: biochemical and functional characterisation

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, γ-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish. Electronic Supplementary Material Supplementary material is available for this article at . The zebrafish MPR 46 sequence data have been submitted to the GenBank database under accession no. DQ089037.     

Differential effect of saturated, monounsaturated, and polyunsaturated fatty acids on alloxan-induced diabetes mellitus

Earlier, we reported that oils rich in omega-3 eicosapentaenoic acid and docosahexaenoic acid and omega-6 gamma-linolenic acid and arachidonic acid prevented the development of alloxan-induced diabetes mellitus in experimental animals. Here we report the results of our studies with pure saturated stearic acid (SA), monounsaturated oleic acid (OA) and omega-6 arachidonic acid (AA) on alloxan-induced diabetes mellitus in Wistar male rats. Prior oral supplementation with AA prevented alloxan-induced diabetes mellitus, whereas both SA and OA were ineffective. Cyclo-oxygenase (COX) and lipoxygenase (LO) inhibitors did not block this protective action of AA against alloxan-induced diabetes, suggesting that both prostaglandins and leukotrienes are not involved, and that AA by itself is effective. Furthermore, AA restored the anti-oxidant status to normal range in various tissues. These results suggest that AA protects pancreatic beta cells against alloxan-induced diabetes in experimental animals by attenuating oxidant stress.     

Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.     

The gastroenteroinsular response to glucose ingestion during postexercise recovery

This study examined gastrointestinal hormone and peptide responses when glucose was ingested after prolonged exercise. Six endurance-trained male athletes ran on a treadmill for 2 h at 60% VO2 max. Immediately after the run, the athletes consumed 75 g of glucose in 250 ml of water (ExGLU) or flavored water as a placebo control (ExPL). On a separate visit, the athletes rested for 2 h and then consumed glucose (ConGLU). During the first 60 min of recovery from exercise alone (ExPL), plasma vasoactive intestinal peptide (VIP), gastrin, and glucagon-like peptide-1 (GLP-1) all increased significantly, whereas glucose, insulin, and gastric inhibitory polypeptide (GIP) were unchanged from the immediate postexercise value. When glucose was ingested after exercise (ExGLU), glucose, insulin, VIP, gastrin, GLP-1, and GIP were all increased (P < 0.01). However, when glucose was ingested after resting for 2 h (ConGLU), VIP levels were unaffected, although glucose, insulin, gastrin, GLP-1, and GIP levels increased (P < 0.05). The plasma glucose response was greater (P < 0.03) and the plasma insulin response lower (P < 0.004) during ExGLU compared with ConGLU. There was a significantly higher (P < 0.01) VIP response during the initial period of recovery in ExGLU than there was with both ExPL and ConGLU. Plasma VIP showed a modest negative correlation with circulating glucose (r = -0.35, P < 0.03) and insulin (r = -0.37, P < 0.03) during the ExGLU recovery period. In summary, when glucose is ingested after prolonged exercise, there is mild insulin resistance and a corresponding rapid transitory increase in plasma VIP. These data suggest that VIP may play an important glucoregulatory role when glucose is ingested during the immediate postexercise recovery period.     

RhoA- and PKC-alpha-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-alpha. Presently, we examined the cross talk between RhoA and PKC-alpha in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-alpha, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-alpha by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-alpha pathways, suggesting cross talk between RhoA and PKC-alpha resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.