Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases.

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake.

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

Accelerated evolution of crotalinae snake venom gland serine proteases.

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

Gastric mucosal protection by OPC-12759, a novel antiulcer compound, in the rat.

OPC-12759, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid, was studied for its efficacy to prevent the gastric mucosal damage induced by several necrotizing agents. Experiments were also performed to elucidate the mechanism of this mucosal protective activity. OPC-12759 dose dependently prevented the formation of mucosal necrosis induced by absolute ethanol, 0.2 N NaOH or 0.6 N HCl. PGE2 was also shown to prevent the gastric mucosal erosion induced by necrotizing agents. The mucosal protective effect of OPC-12759 was completely counteracted by pretreatment with indomethacin while that of PGE2 was not. In addition, OPC-12759 given alone increased the generation of gastric mucosal PGE2-like activity. OPC-12759 dose dependently reduced the volume, acid output and pepsin output of the gastric juice in pylorus-ligated rats. The inhibitory effect of OPC-12759 but not of cimetidine or atropine on gastric secretion was also abolished by concurrent administration of indomethacin. These findings suggest that the mucosal protective effect and antisecretory effect of OPC-12759 presumably result from enhancement of the generation of endogenous PGs.

Potentiation of anti-platelet aggregating activity of cilostazol with vascular endothelial cells.

We developed the new system in which the platelet aggregation was detected turbidimetrically using glass cuvettes which were coated cultured vascular endothelial cells. Using this system, we evaluated the effect of cilostazol, having a selective inhibitory effect on cAMP-PDE, on the ADP-induced platelet aggregation in the presence of endothelial cells. Cilostazol inhibited the platelet aggregation dose-dependently in the presence or absence of endothelial cells. The inhibitory effect of cilostazol on platelet aggregation was potentiated by the presence of endothelial cells, and the slope of the dose-response curves were identified to be as the same between both experiments in the presence and the absence of endothelial cells. The pretreatment of endothelial cells with aspirin reversed the potentiated inhibitory effect of cilostazol on the platelet aggregation with endothelial cells. The amount of 6-keto-prostaglandin F1 alpha accumulated in the cuvette was reduced in this condition. On the other hand, the inhibitory effect of prostaglandin E1 on the platelet aggregation was not potentiated by the presence of endothelial cells. These results suggest that the endothelium-derived prostacyclin plays a role on the potentiation of anti-platelet aggregatory effect of cilostazol.

Characterization, amino acid sequence and evolution of edema-inducing, basic phospholipase A2 from Trimeresurus flavoviridis venom.

Two phospholipases A2 (PLA2s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp49]PLA2) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA2s in Fig. 8), thus belonging to an [Asp49]PLA2 subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the beta-sheet region. On the other hand, PLA-A had beta-Asp-Gly and alpha-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X', whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a beta-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA2 isozymes indicated that PLA-B and PL-X' diverged from PLA2 after branching of [Asp49]PLA2 forms and [Lys49]PLA2 forms.

Retrotransposable CR1-like elements in crotalinae snake genomes.

A part of the 3'-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A2 (PLA2) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA2 isozyme genes, T. flavoviridis PLA2 inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements.

Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Lobular form idiopathic glomerulonephritis with massive subendothelial and paramesangial immune deposits, a three-year follow-up case.

We report a patient with unusual glomerulonephritis. A 24-year-old Japanese female was hospitalized in October 1995 because of nephrotic syndrome. Lobular form glomerulonephritis with mesangial proliferation associated with massive wide-spread accumulation of slightly eosinophilic, periodic acid Schiff-positive amorphous materials in the luminal side of the capillary walls and paramesangial area was observed in the renal biopsy specimen. Immunofluorescent study revealed massive strong staining for IgM and C4 along the capillary walls and in the mesangium. Deposits of IgA, IgG, C3 and fibrinogen were also observed. Electron microscopy showed normal thickness of the capillary basement membrane and a large amount of subendothelial and paramesangial electron dense, finely granular deposits without fibrils or tubular structures. There were no clinical or laboratory findings of systemic diseases, such as systemic lupus erythematosus and cryoglobulinemia. Therefore, we believed that this case involved an unusual idiopathic glomerular disease with massive subendothelial and paramesangial immune deposits. Glomerulonephritis in this patient appeared to be resistant to treatment with corticosteroids and that this glomerulopathy may be a progressive disease as shown during the 3-year observation. Furthermore, our patient had idiopathic hyperprolactinemia and subclinical hypothyroidism. However, the relationship between glomerulonephritis and endocrinopathy in our patient is unknown.

Serum transferrin receptor levels in patients with rheumatoid arthritis are correlated with indicators for anaemia.

To measure serum soluble transferrin receptor (s-TfR) levels in patients with rheumatoid arthritis (RA), sera were obtained from 50 Japanese RA patients and 20 healthy subjects. Both s-TfR and serum erythropoietin (EPO) levels were measured by enzyme-linked immunosorbent assay (ELISA). Routine laboratory tests were also performed, including peripheral blood analysis and determination of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), serum iron levels, total iron-binding capacity (TIBC) and serum ferritin levels. The s-TfR levels in the 50 RA patients (mean +/- SD, 1,801 +/- 512 ng/ml) were significantly higher than those in the 20 control subjects (1,316 +/- 345 ng/ml). There were no differences in the values of s-TfR between men and women in either group, or between RA patients over and under 50 years old. Serum EPO levels in 47 RA patients were as low as 14.0 +/- 10.1 mlU/ml (mean +/- SD), ranging from 3.9 to 58.7 mIU/ml (normal range 2.8-17.2 mlU/ml), unrelated to low haemoglobin concentration. The s-TfR levels in RA patients showed negative correlations with red blood cell count, serum iron level and haemoglobin concentration, and positive correlations with ESR and serum EPO levels. However, there were no correlations between s-TfR level and markers of inflammation such as CRP, platelet count or RF titre. In conclusion, s-TfR level in RA patients could be a marker of erythropoiesis rather than of joint inflammation.

Weber-Christian disease presenting with ocular manifestations.

A Japanese patient with Weber-Christian disease (WCD) presenting with ocular symptoms is reported. Panniculitis in the retrobulbar fat was diagnosed according to the histological findings from biopsy specimens, and improved over a month under steroid administration. Only three patients showing ocular manifestations have previously been reported. Panniculitis was in the late stage and a medium dose of prednisolone was effective in this patient. A biopsy of the orbital fat was useful for diagnosis. However, it is important to recognise that the stage of inflammation varies according to the fat tissue involved by WCD.

[Correlation of serum IgA levels with serum IgG levels, erythrocyte sedimentation rate and platelet counts in 98 patients with rheumatoid arthritis].

Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Polyclonal B cell activation (PBA) is one of immunological abnormalities commonly found in RA patients. We examined serum IgG, IgA, IgM levels in 98 RA patients and compared 31 patients with high serum IgA levels (group B) with 67 patients with normal serum IgA levels (group A) in clinical background. Group B patients had significantly higher mean values of serum IgG levels, erythrocyte sedimentation rate (ESR), and platelet counts than group A. However, there was no correlation between serum IgA levels and X-ray stage, class of ADL or disease duration of RA. These results indicate that high serum IgA levels reflect for disease activity of RA. Serum IgA levels did not correlate with interleukin (IL)-6 levels in 53 RA patients studied. It is speculated that high serum IgA levels might be caused by the following evidences 1) that transforming growth factor (TGF) beta, a known cytokine to increase IgA production by human splenic B cells, gene expression is enhanced in mononuclear cells from synovial fluid and 2) that iron deposition is found in RA synovial and high serum IgA levels are found in iron overload like thalassemia intermedia.

[A case of ANCA positive idiopathic crescentic glomerulonephritis initiated with fever and liver dysfunction].

We studied a case of a 63 year old Japanese man who presented in October, 1994 with general fatigue, low grade fever, micro hematuria and leukocytosis, elevated CRP as well as liver dysfunction. A liver biopsy at that time revealed mild cholangiolitis. Six months later he was admitted because of weight loss, protein urea, and renal failure. At that time he was positive for antineutrophil cytoplasmic antibody(ANCA) with perinuclear staining patter(p-ANCA) done by indirect immunofluorescence. He was also positive for anti-myeloperoxidase antibody(MPO-ANCA) done by ELISA. A renal biopsy showed idiopathic crescentic glomerulonephritis with pauci-immune type(ICGN). Despite therapy with steroids and cyclophosphamide, which improved his subjective symptoms, his renal failure accelerated necessitating hemodialysis which he has been on for over four years. In conclusion, this patient has a rare case of ICGN that presented with liver dysfunction similar to autoimmune hepatitis. Since ANCA has been known to be associated with systemic vasculitides as well as chronic inflammatory diseases(e.g. ICGN, microscopic polyarteritis nodosa, ulcerative colitis or autoimmune liver diseases), both the crescent formation in this patient's glomeruli and cholangiolitis in his liver may have shared the common etiology related to ANCA.

[Anti-myeloperoxidase and anti-lactoferrin antibodies in patients with IgA nephropathy and Henoch-Schönlein purpura].

Antineutrophil cytoplasmic antibodies (ANCA) for two antigens, i.e. myeloperoxidase (MPO) and lactoferrin (LF) in sera from 19 IgA nephropathy (IgAN), 3 adult Henoch-Schönlein purpura (HSP) and 8 child HSP patients were examined by enzyme-linked immunoabsorbent assay (ELISA) for immunoglobulin isotypes. All of child HSP patients showed negative ANCA. On the other hand, one IgAN patient and two adult HSP patients showed weak positivity for IgA class anti-MPO antibody. There was no patients who showed positivity for IgG and IgM class anti-MPO antibody. In anti-LF antibody, one IgAN and one adult HSP showed positivity in IgG class; 2 IgAN and 2 HSP in IgA class and 2 IgAN and one HSP in IgM class. These results indicate that adult HSP patients have higher prevalence of IgA class anti-MPO antibody and anti-LF antibody than IgAN or child HSP.

A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I from bovine testis.

When screening various isoquinolinesulfonamide compounds which we synthesized, CKI-7, N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, was found to have a potent inhibitory action against casein kinase I and a much weaker effect on casein kinase II and other protein kinases. Kinetic analysis indicated that CKI-7 inhibited casein kinase I competitively with respect to ATP and that the Ki values were 8.5 microM for casein kinase I and 70 microM for casein kinase II. An affinity chromatography absorbent was synthesized by coupling CKI-8 (1-(5-chloroisoquinoline-8-sulfonyl], a derivative of CKI-7, to cyanogen bromide-activated Sepharose 4B. Partially purified casein kinase I from bovine testis was subjected to affinity chromatography. Analysis of the purified casein kinase I by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single band with molecular weight 37,000. These newly synthesized compounds, CKI-7 and CKI-8, should serve as useful tools for elucidating the biological significance of casein kinase I-mediated reactions.

Degeneration of zonular fibrils in a case of exfoliation glaucoma.

We examined ultrastructurally zonules of the lens obtained from a patient with exfoliation glaucoma and lens dislocation. There was a great accumulation of exfoliation materials adjacent to and within degenerated zonules, and a gradual transition between zonular fibrils and exfoliation materials was observed. The initial change in the zonular fibrils was a loss of their regularity, next we could see fine granules deposited on degenerated zonular fibrils. A sparse and irregular deposition was found on the surface of the fibrils. Subsequently, the deposition became increased, and densely stained exfoliation materials, which were composed of a single degenerated zonular fibril and fine granules, were formed. Several fibrils accompanied by fine granules clustered together and became exfoliation materials of larger diameter. These observations suggest that degenerated zonular fibrils may develop into exfoliation materials.

Making of a facial perforator map by thermography.

It has been confirmed that thermography is an effective method by which to locate perforators to be used for local flaps. One disadvantage of thermography is its complicated procedure. If it was possible to identify perforators quickly and easily, many operations would be dramatically simplified. The authors' objective was to develop a method of mapping surface perforators using thermography. They pressed a vinyl bag filled with ice water against a test area for 25 seconds and began photographing the area directly after icing. Almost all of the enhanced hot spots appeared for 65 seconds after icing. The number and locations of enhanced perforating vessels varied widely, which they anticipated as a result of differences among individuals, and fluctuations in the measuring environment and the performance of measuring instruments. However, the presence of many perforators is common, and these images are considered to be reliable. Based on their findings, the authors consider the facial perforator map to be accurate and useful. Application of the facial perforator map may simplify many operations and may contribute to making surgery safer and more effective.

ML-9 inhibits the vascular contraction via the inhibition of myosin light chain phosphorylation.

We investigated the effects of a newly synthesized compound, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor of superprecipitation of actomyosin, isometric tension development, and phosphorylation of the 20,000-Da myosin light chain (LC20) in vascular smooth muscle. Superprecipitation of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscles of the rabbit mesenteric artery, ML-9 inhibited the Ca2+-independent contraction provoked by application of trypsin-treated MLCK. In the intact rabbit mesenteric artery, increases in LC20 phosphorylation reached a maximal value of 0.49 mol of Pi/mol of LC20 within 10 sec from a resting value of 0.15 mol of Pi/mol of LC20 and then declined to near the basal level during the maintained isometric force developed in response to 50 mM KCl. Preincubation with 10-30 microM ML-9 for 30 min significantly inhibited both the maximal rate and extent of KCl-induced contraction and the phosphorylation of LC20, in a dose-dependent manner. There was a linear relationship between the initial rate of tension development and the extent of LC20 phosphorylation at 10 sec after stimulation. ML-9 nonspecifically antagonized the contraction induced by various contractile agonists, such as CaCl2, norepinephrine, serotonin, histamine, and angiotensin II. ML-9 dose dependently produced a shift to the right and down, in the dose-response curves, to all the agonists tested. These results suggest that ML-9 inhibits the actin-myosin interaction through the modulation of LC20 phosphorylation via the inhibition of MLCK activity. Thus, ML-9 may be a useful compound for investigating the physiologic role of myosin light chain phosphorylation by MLCK in living cells and tissues as well as in vitro.

Lys-49-phospholipases A2 as active enzyme for beta-arachidonoyl phospholipid bilayer membranes.

Phospholipases A2 containing Lys-49 have been reported to be extremely weak or inactive as enzyme. We have recently shown that basic proteins I and II (BP-I and BP-II), Lys-49-PLA2s isolated from the venom of Trimeresurus flavoviridis (Habu snake), are potent to hydrolyze the arachidonate of 2-arachidonoyl-1-stearoyl-L-3-phosphatidylcholine (ASPC) in bilayer vesicles. In order to ensure such enzymatic activity of Lys-49-PLA2s, two other Lys-49-PLA2s from different snake venoms, myotoxin II (from Bothrops asper) and App-K49 (form Agkistrodon piscivorus piscivorus), were examined. Myotoxin II was found to be very active, even more potent than BP-II, liberating about 80% of arachidonic acid from liposomes. App-K49 was also active (about 50%) for ASPC liposomes. They were very weak or almost inactive for ASPC micelles and monomers. All these Lys-49-PLA2s were inactive for ASPC liposomes in the absence of Ca2+. These results clearly demonstrated that Lys-49-PLA2s are the enzymes to hydrolyze the C2-ester bond of ASPC in bilayer membranes.

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II.

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.