Serum paraoxonase activity decreases in rheumatoid arthritis

OBJECTIVE: To estimate the alterations of paraoxonase 1 (PON1) and high-density lipoprotein (HDL) in rheumatoid arthritis (RA). DESIGN AND METHODS: We investigated the serum enzyme activity and concentration of PON1 and their relationship with serum lipids, high-density lipoprotein (HDL) parameters, and acute phase reactants of serum amyloid A (SAA) and C-reactive protein (CRP) in patients with RA. RESULTS: Serum paraoxonase (PON) activity was significantly decreased in RA patients (n = 64, 131 +/- 53 micro mol/min/L) compared with healthy subjects (n = 155, 164 +/- 59) despite the absence of any difference in serum lipid levels between the two groups. This decrease of serum PON activity in RA patients was found in every genotype (Q/Q, Q/R, R/R) of PON1 at 192 Q/R. There was a different distribution in PON1 Q/R genotypes between RA patients and healthy subjects, and RA patients exhibited less (44%) positive PON1-Q than did the healthy subjects (66%). In a further investigation of age- and gender-matched subgroups of RA (n = 25) and healthy subjects (n = 25), not only serum PON activity, but also lecithin-cholesterol acyltransferase (LCAT) was found to be significantly decreased in RA patients (125 +/- 61 micro mol/min/L, 63.2 +/- 17.2 nmol/ml/hr/37 degrees C) than in healthy subjects (169 +/- 67, 74.7 +/- 19.5), respectively. PON1 and LCAT as well as HDL constituent apolipoprotein (apo) AI and apo AII, were altered significantly in RA patients. CONCLUSIONS: Acute-phase HDL, which is remodeled structurally and functionally in RA, might be less anti-atherogenic due to the impairment of original HDL function. These alterations of HDL in RA patients may explain in part the reported increase in cardiovascular mortality in patients with RA.     

Are there two forms of beta 2-N-acetylglucosaminyltransferase I in rat testicular and epididymal fluids?

The activity of alpha 3-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was detected in rat testicular and cauda epididymal fluids. The GnT I activity of testicular fluid had a pH optimum of 6.0, whereas that of the cauda epididymal fluid was optimal at pH 7.0. The enzyme in testicular fluid had an absolute requirement for either Co2+, or Mn2+, Mg2+ and Ca2+, the activity being stimulated by these cations in the above order, whereas that of cauda epididymal fluid had an absolute requirement for Mn2+ or Ca2+, with Co2+ and Mg2+ being ineffective. The specific activity of GnT I in cauda epididymal fluid was somewhat higher than in testicular fluid. The apparent Km value for alpha 1-3 alpha 1-6mannopentaose of GnT I in the testicular and epididymal fluids was 0.57 and 0.38 mM, respectively. The substrate specificity for both GnT I activities decreased in the following order: alpha1-3 alpha 1-6mannopentaose>alpha1-3 alpha 1-6mannotriose>alpha 1-3mannobiose>alpha 1-6mannobiose. These data suggest that two forms of GnT I exist in the testicular and epididymal fluids.     

1,5-Anhydroglucitol stimulates insulin release in insulinoma cell lines

Concentrations of 1,5-anhydroglucitol (1,5-AG), which is a major circulating polyol, decrease in patients with diabetes mellitus. In both insulinoma-derived RINr and MIN6 cells, 1,5-AG stimulated insulin release within the range of 0.03-0.61 mM in a dose-dependent manner. Insulin release was maximally stimulated by 1,5-AG to levels that reached 25% and 100% greater than that of control (1,5-AG-free group) in RINr and MIN6 cells, respectively. A physiological concentration of 1,5-AG stimulated insulin release after a 5-min incubation and this action was maintained for 60 min. In addition, at approximately 1/200 the concentration of glucose, 1,5-AG had additive action with 20 mM glucose. The action of 1,5-AG on insulin secretion with other types of saccharides and polyol was similarly additive. Mannnoheptulose and diazoxide suppressed the stimulative action of 1,5-AG on insulin release. The secretagogue action of 1,5-AG seemed to be independent on an increase in the intracellular content of cAMP and ATP. These results suggest that 1,5-AG can stimulate insulin secretion through a mechanism that completely differs from that of glucose.     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.     

Possible involvement of optimally phosphorylated L-plastin in activation of superoxide-generating NADPH oxidase

The involvement of protein phosphatases in the activation of superoxide (O2-)- generating enzyme in human neutrophils was examined using calyculin A, an inhibitor of protein phosphatase type 1 and 2A. Calyculin A inhibited the phorbol myristate acetate (PMA)- and opsonized zymosan (OZ)-activated O2- generation by human neutrophils. This inhibitory effect of calyculin A on PMA-activated O2- generation was reversed by the addition of KT5926, a specific inhibitor of myosin light chain kinase and Ca2+/calmodulin-dependent protein kinase II. These results suggest that the addition of calyculin A may cause hyperphosphorylation of some protein(s) that plays a crucial role in the PMA-dependent activation of O2- generating enzyme, and that this protein hyperphosphorylation may be evoked by a KT5926-sensitive kinase or its downstream kinase. Whereas two-dimensional analysis involving 32P revealed that calyculin A caused the hyperphosphorylation of many proteins, KT5926 mainly reduced the calyculin A-induced hyperphosphorylation of a 67 kDa protein in activated neutrophils, suggesting that the hyperphosphorylation of the 67 kDa protein might inhibit the PMA-dependent activation of NADPH oxidase. The 67 kDa cytosolic protein was moderately phosphorylated on the addition of PMA. On the other hand, in the absence of calyculin A, KT5926 inhibited both PMA-induced O2- generation and phosphorylation of the 67 kDa protein. Amino acid sequence analysis of peptides derived from the 67 kDa protein revealed that the 67 kDa protein was identical to L-plastin, an actin-bundling protein. We conclude that optimally phosphorylated L-plastin may play some crucial role in the activation of NADPH oxidase.     

Cross-talk between the pathways leading to the induction of apoptosis and the secretion of tumor necrosis factor-alpha in ricin-treated RAW 264.7 cells

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-alpha (TNF-alpha) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-alpha from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-alpha, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-alpha and apoptosis. Time course analysis revealed that the activation of p38 MAP kinase started prior to the induction of TNF-alpha release and apoptosis. Since the activation of p38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-alpha, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p38 MAP kinase, which in turn leads to TNF-alpha release and apoptosis.