Cytokeratin19 induced by HER2/ERK binds and stabilizes HER2 on cell membranes.

Cytokeratin19 (KRT19) is widely used as a biomarker for the detection of disseminated tumors. Using an LC-MS/MS proteomics approach, we found that KRT19 was upregulated in HER2-overexpressing cells and tissues. KRT19 expression was induced by HER2-downstream ERK at the transcriptional level. Another HER2-downstream kinase, Akt, was found to phosphorylate KRT19 on Ser35 and induce membrane translocation of KRT19 and remodeling of KRT19 from filamentous to granulous form. KRT19 phosphorylated by Akt could bind HER2 on the plasma membrane and stabilized HER2 via inhibition of proteasome-mediated degradation of HER2. Silencing of KRT19 by shRNA resulted in increased ubiquitination and destabilization of HER2. Moreover, treatment of KRT19 antibody resulted in downregulation of HER2 and reduced cell viability. These data provide a new rationale for targeting HER2-positive breast cancers.

Sepsis affects most routine and cell population data (CPD) obtained using the Sysmex XN-2000 blood cell analyzer: neutrophil-related CPD NE-SFL and NE-WY provide useful information for detecting sepsis.

INTRODUCTION: The Sysmex XN-2000 analyzer can assess 36 routine and 57 cell population data (CPD) items. In this study, we evaluated these items as sepsis biomarkers. METHODS: We enrolled 280 normal control (NC) and 130 sepsis patients. The sepsis patients were classified as uncomplicated or complicated sepsis. Routine and CPD items were determined, and the results were compared at between the NC and sepsis groups, uncomplicated and complicated sepsis groups, and survivors and nonsurvivors. RESULTS: For the detection of sepsis, CPD items NE-SFL [defined as the fluorescent light intensity of the neutrophil area on the WDF (white blood cell differential) scattergram] and NE-WY (defined as the fluorescent light distribution width of the neutrophil area on the WDF scattergram) showed comparative or higher AUC of 0.909 and 0.905, respectively, when compared with routine items such as hematocrit, hemoglobin, RBC, RDW, immature granulocytes count, lymphocytes count, and neutrophils count. For the discrimination of sepsis severity, only platelet-related items showed higher AUC (0.723 - 0.748) than lactic acid (0.695). For the prediction of 28-day mortality, only CV and SD of RDW showed higher AUC (0.766 and 0.732 each) than lactic acid (0.712). CONCLUSIONS: Sepsis patients demonstrated significant changes in routine and CPD items related to RBC, neutrophils, lymphocytes, and platelets when compared to NCs. Increase in CPD items NE-SFL and NE-WY, which may indicate neutrophil immaturity or activation, could be useful for the detection of sepsis patients, in conjunction with currently used surrogate sepsis biomarkers. However, these items did not efficiently contribute to the discrimination of sepsis severity or predict mortality.

Antimutagenicity and induction of anticarcinogenic phase II enzymes by basidiomycetes.

Extracts from Phellinus linteus, Phellinus igniarius, and Agrocybe cylindracea have been tested for their antimutagenic properties against direct-acting mutagens [4-nitro-o-phenylenediamine (NPD) and sodium azide (NaN(3))] and indirect-acting mutagens [2-aminofluorene (2-AF) and benzo[a]pyrene (B[a]P)], using the Salmonella typhimurium tester strains TA 98 and TA 100. In addition, the chemopreventive potentials of these extracts to induce NAD(P)H:quinone oxidoreductase (QR) and glutathione S-transferase (GST) activities and glutathione (GSH) level extracts from the filtrate of the cultured broth of P. linteus, polysaccharide extracts from the cultured broth (PI I) and mycelia (PI II) and water extract of fruiting bodies (PI II) of P. igniarius, and polysaccharide extracts from the cultured broth (AC I) and mycelia (AC II) of A. cylindracea showed inhibitory effects on the mutagenic activities induced by the direct-acting mutagens, NPD and NaN(3), and the indirect-acting mutagens, 2-AF and B[a]P. QR was induced with PI I, PI II, AC I, and AC II, and GST activity was induced with PL I, PL II, PI I, PI II, PI III and AC I in murine Hepa1c1c7 cell culture. In addition, PL I, PL II, PI I, PI II, PI III and AC II increased glutathione level. These results suggest that P. linteus, P. igniarius, and A. cylindracea have antimutagenic activities and may play a role in the prevention of cancer by inducing QR and GST activities and increasing GSH level.

Cytotoxic activities of acetoxyscirpenediol and ergosterol peroxide from Paecilomyces tenuipes.

Paecilomyces tenuipes is one of the famous Chinese medicinal entomopathogenic fungi that parasites in the lavae of silkworm. Two cytotoxic components were isolated from methanolic extract of the carpophores of this fungus that was cultivated artificially. Spectral analyses of the cytotoxic components showed that they were known ergosterol peroxide (5alpha,8alpha-epidioxy-24(R)-methylcholesta-6,22-dien-3beta-ol) and acetoxyscirpenediol (4beta-acetoxyscirpene-3alpha,15-diol) that were isolated for the first time from this fungus. The 50% inhibitory concentrations (IC50) of ergosterol peroxide against human gastric tumor cell line (SNU-1), human hepatoma cell line (SNU-354), human colorectal tumor cell line (SNU-C4) and murine sarcoma-180 were 18.7, 158.2, 84.6 and 74.1 microM, respectively. The IC50 values of acetoxyscirpenediol against SNU-1, SNU-C4, SNU-354 and sarcoma-180 were 1.2,4.0, 2.2 and 1.9 microM, respectively. Cytotoxic activities of acetoxyscirpenediol were about 4.0-6.6 times stronger than those of cisplatin which is currently used clinically for cancer patients.

Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin.

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.

Bombycis corpus extract prevents amyloid-beta-induced cytotoxicity and protects superoxide dismutase activity in cultured rat astrocytes.

Bombycis corpus(BC) or Bombyx Batryticatus, a batryticated silkworm and white-stiff silkworm, is a drug consisting of the dried larva of silkworm, Mobyz mori L., dead and stiffened due to the infection of Beauveria (Bals.) Vuill. In the present study, we have examined the protective effect of the water extracts against Amyloid- beta(A beta) 25-35 peptide-induced cytotoxicity by microscopic observation and LDH assay, and its action on antioxidative enzymes using cultured astrocyte cells. A beta 25-35-induced cell death was protected by the application of water extract of BC in a dose-dependent manner, and concentrations of 10(-6)to 10(-7)g ml(-1)showed a significant effect compared to exposure of A beta 25-35 alone. When antioxidative enzyme activities such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S transferase (GST) were assayed after A beta 25-35 treatment, most enzyme activities were decreased in a similar fashion. BC treatment of A beta 25-35-treated astrocytes did not affect the enzyme activities of catalase, GSH-Px and GST. However, only SOD activity was enhanced by BC treatment and this may result from the potentiation of the antioxidative ability of BC. The protective effect of BC against cytotoxicity induced by Abeta 25-35 strongly indicates that BC could be a protective agent for free radical generating compounds, and that Abeta 25-35 is not only a potent lipid peroxide inducer, but can also cause changes in antioxidative enzymes. From the results, it was concluded that BC has a protective effect against Abeta -induced cytotoxicity in cultured astrocyte cells through the inhibition of lipid peroxidation and protection of antioxidative enzymes.

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase.

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.

Cinnamaldehyde inhibits lymphocyte proliferation and modulates T-cell differentiation.

Two kinds of cinnamaldehyde derivative, 2'-hydroxycinnamaldehyde (HCA) and 2'-benzoxy-cinnamaldehyde (BCA), were studied for their immunomodulatory effects. These compounds were screened as anticancer drug candidates from stem bark of Cinnamomum cassia for their inhibitory effect on farnesyl protein transferase activity. Ras activation, which is accompanied with its farnesylation, has been known to be important in immune cell activation as well as in carcinogenesis. Treatment of these cinnamaldehydes to mouse splenocyte cultures induced suppression of lymphoproliferation following both Con A and LPS stimulation in a dose-dependent manner. A dose of I microM of HCA and BCA inhibited the Con A-stimulated proliferation by 69% and 60%, and the LPS-induced proliferation by 29% and 21%, respectively. However, the proliferation induced by PMA plus ionomycin was affected by neither HCA nor BCA treatment. Decreased levels of antibody production by HCA or BCA treatment were observed in both SRBC-immunized mice and LPS-stimulated splenocyte cultures. The exposure of thymocytes to HCA or BCA for 48 h accelerated T-cell differentiation from CD4 and CD8 double positive cells to CD4 or CD8 single positive cells. The inhibitory effect of cinnamaldehyde on lymphoproliferation was specific to the early phase of cell activation, showing the strongest inhibition of Con A- or LPS-stimulated proliferation when added concomitantly with the mitogens. In addition, the treatment of HCA and BCA to splenocyte cultures attenuated the Con A-triggered progression of cell cycle at G1 phase with no inhibition of S to G2/M phase transition. Although cinnamaldehyde treatment had no effect on the IL-2 production by splenocyte cultures stimulated with Con A, it inhibited markedly and dose-dependently the expression of IL-2Ralpha and interferon-gamma. Taken together, the results in this study suggest both HCA and BCA inhibit the lymphoproliferation and induce a T-cell differentiation through the blockade of early steps in signaling pathway leading to cell growth.

A visual membrane immunoassay for the detection of methamphetamine using an enzyme-labeled tracer derived from methamphetamine and amphetamine.

A visual membrane enzyme immunoassay is described for the measurement of methamphetamine in urine. To increase assay sensitivity, tracers with chemically similar structures were cross-checked with the antibodies to determine their influence on the antibody binding. Tracers of horseradish peroxidase-labeled methamphetamine (MA-HRP) and amphetamine (A-HRP) derivatives were prepared for this purpose. Significant differences in antibody specificity were found between the two tracers. Based on the results of this study, a pair of an antibody and a tracer was selected and a membrane enzyme immunoassay (EIA) was developed utilizing the competitive binding between methamphetamine and the drug-HRP tracer. UltraBind membrane (0.45 micron) was used as the solid matrix to which the antibody was attached. Using diaminobenzidine substrate with Co2+ ion, a stable grey color appeared on the surface of membrane for MA-negative urine samples. No color appeared for MA-positive urine with a cut-off level of 0.8 ppm.

Production and characterization of monoclonal antibody that simultaneously recognizes methamphetamine and its major metabolite.

A series of monoclonal antibodies (mAbs) that react with methamphetamine-bovine serum albumin (MA-BSA) were established by intrasplenic immunization method. Among established 36 clones, two typical mAbs, designated NK-1 and NK-2, were described. The inhibition assay of enzyme-linked immunosorbent assay (ELISA) analysis using methamphetamine analogs indicated that NK-1 showed considerable reactivity not only MA-BSA but also methamphetamine and its major metabolite, para-hydroxymethamphetamine (p-hydroxymethamphetamine). The cross-reactivity between NK-1 and the methamphetamine analogs with modified alkyl side chain, indicates that methyl groups of R5 and R7 in the methamphetamine molecules are important for the maximum affinity. The length of alkyl side chain on methamphetamine significantly affected the binding affinity of NK-1. The results may suggest that NK-1 will recognize not only methamphetamine but also the bridge part of the methamphetamine that binds the methamphetamine molecules to a carrier protein.

Carpal and tarsal osteolysis: an MRI, angiographic and histopathologic study.

The case of a 13-year-old girl with striking carpal and tarsal osteolysis (sporadic occurrence) is reported. MRI confirmed the total absence of carpal bones and medial tarsal bones. Dense fibrocollagenous tissue replaced the spaces left by the resorbed bones. Arteriography showed occlusion of the radial artery at the level of the physis of the distal radius with increased tortuosity of the ulnar artery. There was no major vascular occlusion in the foot except for some indistinct and blurred tarsal branches of the anterior tibial artery.

Effective induction of anti-phospholipid and anticoagulant antibodies in normal mouse.

Anti-phosphatidylserine (anti-PS) antibodies of the IgG isotype in the serum of BALB/c mouse were effectively induced by the intrasplenic immunization of phosphatidylserine (PS), while only a slight increase of the titer was observed when the antigen was injected intravenously. The serum antibodies cross-reacted extensively with other acidic phospholipids, but not with phosphatidylcholine (PC). A remarkable frequency of anti-PS mAbs of the IgG isotype was also observed even when mAbs were produced 10 days after the intrasplenic injection of the antigen. Reactivities of the fifteen mAbs, which had been established from BALB/c mice by the intrasplenic immunization with PS, were further analyzed. Among the fifteen mAbs examined, thirteen cross-reacted with ssDNA and two bound to dsDNA. Seven mAbs had lupus anticoagulant activity and four bound to cultured human umbilical vein endothelial cells (HUVECs). No significant correlation was found among phospholipid specificities, anti-DNA, anticoagulant, and HUVEC binding activities. One mAb of the IgG2b subclass, named PSG3, which had been produced 10 days after the immunization, was shown to have a strong lupus anticoagulant, dsDNA binding, and HUVEC binding activities.

Anti-idiotypic antibody identifies the structural similarity between the phosphatidylcholine-specific monoclonal antibody and phosphatidylcholine-specific lipid transfer protein.

The polyclonal anti-idiotypic antibody (Anti-Id) has been raised against a monoclonal antibody (mAb) that specifically binds to phosphatidylcholine (PC). The anti-Id bound strongly to PC-specific mAbs, but not to the other mAbs that bind to phosphatidylserine, indicating that the anti-Id recognizes the cross-reactive idiotopes expressed on the PC-specific mAbs. The anti-Id also showed an extensive cross-reaction with the PC-specific lipid transfer protein isolated from bovine liver and inhibited the lipid transfer activity of the protein. These results strongly suggest that the anti-Id recognizes a common structure shared between PC-specific mAbs and the PC-specific lipid transfer protein.

Production and characterization of monoclonal antibodies that specifically bind to phosphatidylcholine.

A series of monoclonal antibodies (mAbs) that react with phosphatidylcholine (PC) were established. All mAbs were highly specific to PC and no cross-reaction with other phospholipids were observed. The results obtained with two typical monoclonal antibodies, JE-1 and JE-8, were described. The analysis using synthetic PC analogs with modified polar head groups showed that the methyl groups on the quaternary nitrogen of the choline moiety were important for the binding. Each mAbs showed distinct acyl chain specificities of the PC molecules, and JE-1 showed considerable reactivity with PC with saturated fatty acids, whereas JE-8 could not react with the PC. Both mAbs bound to PC with unsaturated fatty acids, but showed distinct reactivity profiles. Both mAbs reacted only weakly with water-soluble haptens such as phosphorylcholine and L-alpha-glycerophosphocholine, suggesting that the hydrophobic moiety of the PC molecule is important for the maximum affinity. The interaction between the mAbs and the hydrophobic moieties of PC molecules was further studied by analyzing the effect of the mAbs on the activities of phospholipase A2 and phospholipase C. JE-1 inhibited both enzyme activities, while JE-8 inhibited only the phospholipase C activity, indicating that JE-1 interacts more thoroughly with the hydrophobic region of the PC molecule than JE-8 does.

Effective production of monoclonal antibodies against phosphatidylserine: stereo-specific recognition of phosphatidylserine by monoclonal antibody.

A system based on the direct immunization of phospholipid Ag into mouse spleen has been used to produce mAb against phosphatidylserine (PS). mAb that bind to PS but not to phosphatidylcholine were selected. Remarkable frequency of the production of mAb against PS was observed with the immunization protocol. The mAb exhibited three distinct reactivity profiles ranging from highly specific to broadly cross-reactive. Among 61 hybridomas, 15 mAb were established for further analysis. The reactivities of three typical mAb, designated PS4A7, PS3A, and PSC8, are described. PS4A7 is highly specific to PS and no cross-reaction with other acidic phospholipids was observed. In the experiments using PS derivatives with a modified polar head group, PS4A7 was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS) but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine or 1,2-diacyl-sn-glycero-3-phospho-L-homoserine, indicating that the antibody recognizes the stereo-specific configuration of serine residue in PS. PS3A binds to both PS and phosphatidylethanolamine, whereas no cross-reaction with other acidic phospholipids was observed. The analysis using the derivatives of PS and phosphatidylethanolamine shows that the antibody recognizes the amino group of the phospholipid Ag and cannot distinguish the conformational structure of serine residue in PS. PSC8 represents the family of mAb that cross-react considerably with other acidic phospholipids.