Crab meat is a valuable source of proteins and functional lipids and it is widely consumed worldwide. However, the prevalence of crab allergy has increased over the past few years. In order to understand crab allergy better, it is necessary to identify crab allergens. The aim of the present study was to compare the IgEbinding proteins of raw and cooked extracts of mud crab (Scylla serrata). Raw and cooked extracts of the mud crab were prepared. Protein profiles and IgE reactivity patterns were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting using sera from 21 skin prick test (SPT) positive patients. In SDS-PAGE, 20 protein bands (12 to 250 kDa) were observed in the raw extract while the cooked extract demonstrated fewer bands. Protein bands between 40 to 250 kDa were sensitive to heat denaturation and no longer observed in the cooked extract. In immunoblotting experiments, raw and cooked extracts demonstrated 11 and 4 IgE-binding proteins, respectively, with molecular weights of between 23 and 250 kDa. A heat-resistant 36 kDa protein, corresponding to crab tropomyosin was identified as the major allergen of both extracts. In addition, a 41 kDa heat-sensitive protein believed to be arginine kinase was shown to be a major allergen of the raw extract. Other minor allergens were also observed at various molecular weights.
Arginine Kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

Hypernatremia is a rare cause of rhabdomyolysis. Here, we report a case of hypernatremia-induced rhabdomyolysis in a patient with meningioma involving the pituitary gland. A 61-year-old male was admitted for decreased mentality and poor oral intake. He had undergone an operation for meningioma 10 years prior. At admission, he appeared lethargic and severely dehydrated with an initial sodium level of 178 mEq/L. Hypernatremia remained persistent despite massive hydration and the serum creatine phosphokinase level was 18,047 U/L after 3 days. Bone scintigraphy also showed findings consistent with rhabdomyolysis. Brain magnetic resonance imaging revealed extensive masses involving the pituitary gland and an intranasal biopsy confirmed meningioma. Polyuria, and low anti-diuretic hormone levels supported the diagnosis of central diabetes insipidus-induced hypernatremia. Desmopressin was administered intranasally and the patient's serum sodium and muscle enzyme levels were normalized.
Biopsy ; Brain ; Creatine Kinase ; Deamino Arginine Vasopressin ; Diabetes Insipidus ; Diagnosis ; Humans ; Hypernatremia ; Magnetic Resonance Imaging ; Male ; Meningioma* ; Middle Aged ; Pituitary Gland* ; Polyuria ; Radionuclide Imaging ; Rhabdomyolysis* ; Sodium

OBJECTIVETo compare the protective effects of tetramethylpyrazine (TMP) alone and TMP and L-arginine (TMP-LA) combination on rats with acute myocardial infarction (AMI), and to explore its mechanism.

METHODSThe rat model of AMI was established by via caudal vein injection of pituitrin. Experimental animal groups of normal, model, TMP treated and TMP-LA (via abdominal cavity and caudal vein respectively) treated groups were established. Expression of P- and E-selectin, serum creatine phosphokinase (CK) and troponin T (TnT), and marrow peroxidase (MPO) concentration in myocardial tissue were determined by immunohistochemical stain.

RESULTSAs compared with the normal group, serum CK and TnT level, and MPO concentration in myocardial tissue were significantly higher in the model group (P < 0.01), with P- and E-selectin significantly up-regulated (P < 0.01). As compared with the model group, the above-mentioned parameters in the TMP or TMP-LA treated group was significantly lower (P < 0.05).

CONCLUSIONCombined use of TMP and LA showed obvious synergism in treating AMI, by way of multi-link inhibition on expression of adhesive factors and decrease of leucocyte infiltration.

Animals ; Arginine ; pharmacology ; Creatine Kinase ; blood ; Drug Synergism ; Female ; Male ; Myocardial Infarction ; drug therapy ; enzymology ; P-Selectin ; blood ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Troponin T ; blood

In this study, the effect of phorbol ester on the synthesis of nitric oxide(NO) in murine microglial cells was examined. Phorbol 12-myristate 13-acetate(PMA), a protein kinase C(PKC) activator, alone had no effect, whereas PMA with recombinant interferon-gamma(rIFN-gamma) synergistically increased NO synthesis in murine microglial cells. The maximal effect of PMA in the increase of NO synthesis always fit with the range for rull activation of PKC in these cells. The increase of NO synthesis was reflected as increased amount of inducible NO synthase(iNOS) mRNA by Northern blotting. Treatment of PKC inhibitors such as staurosporine(STSN) or polymxin B decreased rIFN-gamma plus PMA-stimulated NO synthesis. Further, prolonged incubation of the cells with PMA, which down regulate PKC activity, abolished synergistic cooperative effect with IFN-gamma. N(G)-monomethyl-L arginine monohydrate(NGMMA), an analogue of L-arginine, and arginase inhibited rIFN-gamma plus PMA-induced NO production in murine microglial cells. On the basis of these observations we conclude that PKC might not be involved in the expression of iNOS, but instead, might be involved in the post-transcriptional modification of iNOS mRNA.
Arginase ; Arginine ; Astrocytes ; Blotting, Northern ; Microglia ; Myristic Acid* ; Nitric Oxide Synthase Type II ; Nitric Oxide* ; Protein Kinase C ; Protein Kinases ; RNA, Messenger

Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27(Kip1), cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH(2))(5), D-Ile(2), Ile(4), Arg8]-vasopressin (0.1 μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27(Kip1), increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G(0)/G(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.
Animals ; Antidiuretic Hormone Receptor Antagonists ; pharmacology ; Arginine Vasopressin ; pharmacology ; Cell Cycle ; Cell Cycle Proteins ; metabolism ; Cell Proliferation ; Fibroblasts ; cytology ; drug effects ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocardium ; cytology ; Phosphorylation ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology

BACKGROUND: Arginine vasopressin (AVP) is widely used as a vasopressor agent. Some recent studies have suggested that AVP may exert an immunomodulatory effect. However, the mechanism about the anti-inflammatory effect of AVP is not well known. We investigated the effect of AVP on the ihibitor of kappa B (IkappaBalpha)/nuclear factor-kappa B (NF-kappaB) pathway in RAW 264.7 cells. METHODS: Cultured RAW 264.7 cells were pretreated with AVP and stimulated with lipopolysaccharide (LPS). To evaluate the effect of AVP on inflammatory cytokines, the concentration of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by an enzyme-linked immunosorbent assay technique. The expression of IkappaBalpha and nuclear translocation of NF-kappaB p65 were measured by Western blotting, and IkappaB kinase (IKK) activity was analyzed by an in vitro immune complex kinase assay. To confirm the AVP effect on IkappaBalpha/NF-kappaB cascade and via V2 receptor, we added tolvaptan (V2 receptor antagonist) after AVP pretreatment. RESULTS: The increase of IL-6 and TNF-alpha in LPS-stimulated RAW 264.7 cells was suppressed by a treatment with AVP. Pretreatment of AVP inhibited increasing of IKK activity and IkappaBalpha degradation induced by LPS in RAW 264.7 cells. Furthermore, LPS induced and NF-kappaB transcription was inhibited by AVP pretreatment. The observed changes in IKK activity, IkappaBalpha degradation and NF-kappaB transcription by AVP was abolished by tolvaptan treatment. CONCLUSIONS: Our results suggest that AVP showed anti-inflammatory effect on LPS-induced IkappaBalpha/NF-kappaB cascade in mouse macrophages via V2 receptors.
Animals ; Antigen-Antibody Complex ; Arginine Vasopressin ; Blotting, Western ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; I-kappa B Kinase ; Interleukin-6 ; Macrophages ; Mice ; NF-kappa B ; Phosphotransferases ; Receptors, Vasopressin ; Tumor Necrosis Factor-alpha

A family with paramyotonia congenita (PC) is presented. At least 10 family members were affected in an autosomal dominant inheritance pattern. The proband had cold-sensitive muscle stiffness, paradoxical myotonia, and intermittent muscle weakness since childhood. The serum level of creatine kinase was mildly elevated and short exercise test with cooling revealed a drastic reduction of compound muscle action potentials with repetitive discharges. Muscle biopsy revealed marked variation in the fiber size and increased internal nuclei. The molecular biological study revealed a common missense mutation (Arg1448Cys) at the voltage-gated sodium channel gene (SCN4A). The repetitive CMAP discharges during short exercise test with cooling observed in the proband has not been reported previously. This observation needs to be confirmed among PC patients with different mutations. This is the first report on a PC family confirmed by the molecular biological technique in Korea.
Adult ; Arginine/*chemistry ; Cell Nucleus/metabolism ; Creatine Kinase/blood ; Cysteine/*chemistry ; DNA Mutational Analysis ; Exercise ; Female ; Humans ; Korea ; Male ; *Mutation, Missense ; Myotonic Disorders/*genetics ; Pedigree ; Phenotype ; Sodium Channels/*genetics/metabolism

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. MATERIALS AND METHODS: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. RESULTS: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. CONCLUSION: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.
Animals ; Aorta ; Arginase* ; Arginine ; Blotting, Western ; Bromodeoxyuridine ; Cell Proliferation* ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Lipoproteins ; Luminescence ; Membranes ; Muscle, Smooth, Vascular* ; NADP* ; NADPH Oxidase* ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Rats ; Reactive Oxygen Species ; Superoxides