MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells.

BACKGROUND: Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood. METHODS: Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA). RESULTS: We showed that several breast cancer cell lines have a small population of CD24(-/low)/CD44(+) cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24(-/low)/CD44(+) cell populations are enriched for genes involved in transforming growth factor-beta, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-kappaB (NF-kappaB) activity in CD24(-/low)/CD44(+) cells, which was previously unrecognised. In addition, NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24(-/low)/CD44(+) cells in vivo. CONCLUSION: Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.

The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development.

The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.

Unique role of SNT-2/FRS2beta/FRS3 docking/adaptor protein for negative regulation in EGF receptor tyrosine kinase signaling pathways.

The membrane-linked docking protein SNT-2/FRS2beta/FRS3 becomes tyrosine phosphorylated in response to fibroblast growth factors (FGFs) and neurotrophins and serves as a platform for recruitment of multiple signaling proteins, including Grb2 and Shp2, to FGF receptors or neurotrophin receptors. We previously reported that SNT-2 is not tyrosine phosphorylated significantly in response to epidermal growth factor (EGF) but that it inhibits ERK activation via EGF stimulation by forming a complex with ERK2. In the present report, we show that expression of SNT-2 suppressed EGF-induced cell transformation and proliferation, and expression level of SNT-2 is downregulated in cancer. The activities of the major signaling molecules in EGF receptor (EGFR) signal transduction pathways, including autophosphorylation of EGFR, were attenuated in cells expressing SNT-2 but not in cells expressing SNT-2 mutants lacking the ERK2-binding domain. Furthermore, SNT-2 constitutively bound to EGFR through the phosphotyrosine binding (PTB) domain both with and without EGF stimulation. Treatment of cells with MEK inhibitor U0126 partially restored the phosphorylation levels of MEK and EGFR in cells expressing SNT-2. On the basis of these findings, we propose a novel mechanism of negative control of EGFR tyrosine kinase activity with SNT-2 by recruiting ERK2, which is the site of negative-feedback loop from ERK, ultimately leading to inhibition of EGF-induced cell transformation and proliferation.

Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation.

Epidermal growth factor (EGF) induces tyrosine phosphorylation of the Shc adapter protein, which plays an important role in EGF-stimulated mitogenesis. Shc stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of Shc, at Y239 and Y240. To define the Shc pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of Shc. We constructed wild-type (WT) or mutant Shc cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F Shc, but not Y317F or Y239/240F Shc, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F Shc or Y239/240F Shc decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F. Although cells expressing the Y317F Shc hardly activated MAPK in response to EGF, cells expressing the Y239/240F Shc fully activated MAPK. In contrast, Y239/240F Shc, but not Y317F Shc, reduced the EGF-induced c-myc message. These results suggest that Shc activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.

DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane.

CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Addiction to the IGF2-ID1-IGF2 circuit for maintenance of the breast cancer stem-like cells.

The transcription factor nuclear factor-κB (NF-κB) has important roles for tumorigenesis, but how it regulates cancer stem cells (CSCs) remains largely unclear. We identified insulin-like growth factor 2 (IGF2) is a key target of NF-κB activated by HER2/HER3 signaling to form tumor spheres in breast cancer cells. The IGF2 receptor, IGF1 R, was expressed at high levels in CSC-enriched populations in primary breast cancer cells. Moreover, IGF2-PI3K (IGF2-phosphatidyl inositol 3 kinase) signaling induced expression of a stemness transcription factor, inhibitor of DNA-binding 1 (ID1), and IGF2 itself. ID1 knockdown greatly reduced IGF2 expression, and tumor sphere formation. Finally, treatment with anti-IGF1/2 antibodies blocked tumorigenesis derived from the IGF1Rhigh CSC-enriched population in a patient-derived xenograft model. Thus, NF-κB may trigger IGF2-ID1-IGF2-positive feedback circuits that allow cancer stem-like cells to appear. Then, they may become addicted to the circuits. As the circuits are the Achilles' heels of CSCs, it will be critical to break them for eradication of CSCs.

Novel approach on the risk assessment of oxidized fats and oils for perspectives of food safety and quality. I. Oxidized fats and oils induces neurotoxicity relating pica behavior and hypoactivity.

Food poisoning caused by deteriorated fat and oil in instant noodles was first reported in Japan approximately 40 years ago. In these cases, many people developed neurotoxic symptoms such as emesis and discomfort. The degree of oxidation of the fat and oil in the instant noodles that induced food poisoning was at least 100 meq/kg in peroxide value (PV). No general toxicity studies with animals, however, have examined the toxicity of fat and oil oxidized to that extent. In this study, pica behavior, a behavior characterized by eating a nonfood material such as kaolin and that relates to the degree of discomfort in animals, and alterations of locomotor activity of rats eating deteriorated fat and oil were measured. The groups fed fat and oil with at least 138.5 meq/kg PV consumed significantly more kaolin compared to the control group. Furthermore, rats that ate deteriorated fat and oil with at least 107.2 meq/kg PV had significantly decreased locomotor activity compared to control rats. These phenomena suggest that oxidized fat and oil with at least 100 meq/kg PV induce neurotoxicity. The toxicity of oxidized fat and oil has only been addressed using general toxicity tests, but the present results reveal the importance of evaluating toxicity by using other measures.

Inner segment ellipsoid band length is a prognostic factor in retinitis pigmentosa associated with EYS mutations: 5-year observation of retinal structure.

PurposeTo evaluate whether the length of the inner segment ellipsoid (ISe) band can be used as a prognostic factor for disease course in retinitis pigmentosa (RP) patients with EYS mutations by observation over a period of 5 years.MethodsTwelve RP patients with EYS mutations were studied. The horizontal and vertical ISe length of the right eye was manually measured at five time points annually, using spectral domain optical coherence tomography. A regression line through the five points from baseline to the final measurement was drawn and the ratio of the length (%) at each point to the baseline length was calculated; the slope was defined as the rate of ISe shortening (%/year). The correlation between the rate of ISe shortening and age, visual acuity, and mean deviation (MD) value were evaluated. The intraclass correlation coefficient (ICC) for the measurements was calculated.ResultsThe mean rate of ISe shortening was -4.65±2.89% per year and the decline was statistically significant. The rate of shortening was significantly negatively correlated with the baseline length (P=0.046, r=0.58), but not with the baseline age, visual acuity, and MD value. The ICC (2, 1) was 0.999.ConclusionsISe of all RP patients with EYS mutations shortened during the 5 years of annual observation. The measurement of the length of ISe is a simple and convenient method with high repeatability, and the length is a sensitive prognostic factor for the rate of ISe shortening in RP patients with EYS mutations.

A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF.

Flt-1 (fms-like tyrosine kinase-1), a receptor-type tyrosine kinase of sharing similar features with two other flt-family encoded proteins KDR/Flk-1 and Flt-4, has been recently identified as a receptor for Vascular Endothelial Growth Factor (VEGF) known to induce the proliferation of vascular endothelial cells. In this study, we demonstrate that Flt-1 encodes for a 180 kDa glycoprotein, binds VEGF with high affinity, undergoes autophosphorylation but does not generate any mitogenic response in transfected NIH3T3 fibroblasts. Interestingly, the immediate early gene c-myc was not induced, whereas the c-fos was induced very weakly in Flt-1 expressing NIH3T3 cells. A comparative analysis of the Flt-1 signal cascade in the environment of endothelial cells with that of Flt-1 expressing NIH3T3 cells showed that VEGF induced phosphorylation of PLC gamma and GAP complex on tyrosine in both type of cells. However, a strong activation of MAP kinases was observed only in endothelial cells. Further, different from many other receptor tyrosine kinases, tyrosine phosphorylation of Shc protein, an important adaptor for signal transduction from many receptor kinases, was very weak in both Flt-1-NIH3T3 cells and endothelial cells. These results suggest that Flt-1 kinase utilizes a unique signal transduction system in endothelial cells, and the activation of the Flt-1 kinase is insufficient to trigger a mitogenic response in NIH3T3 fibroblasts.

The SH2 domain of Shc suppresses EGF-induced mitogenesis in a dominant negative manner.

Recently, we have shown that an EGF-R-mutant lacking the autophosphorylation sites phosphorylates Shc and retains mitogenic activity. In this report, we have shown that in these cells, in response to EGF, Ras is fully activated with formation of the tyrosine-phosphorylated Shc-Grb2-mSOS complex without the receptor. This pointed out the importance of Shc in EGF-induced Ras activation. To investigate the mechanism of tyrosine phosphorylation of Shc by EGF-R, we carried out in vitro kinase assays using immunoprecipitated EGF-R and bacterially-expressed Shc proteins as substrates. The EGF-R phosphorylated Shc, but not the Shc SH2 mutant, lacking binding ability for phosphotyrosine. This suggests that intact Shc SH2 is essential for the full-length Shc to become phosphorylated, probably by inducing a conformational change in Shc. Thus a Shc SH2 peptide may inhibit competitively Shc phosphorylation. We microinjected the Shc SH2 domain into NIH3T3 cells overexpressing the EGF-R. Microinjected Shc SH2 greatly suppressed EGF-induced DNA synthesis. But microinjection of neither the Shc SH2 mutant nor PLC-gamma 1 SH2 had any effect. This suppressing effect was rescued by comicroinjection of the full-length Shc, suggesting Shc SH2 specifically suppressed the Shc pathway. Thus we concluded Shc phosphorylation is crucial, whereas receptor autophosphorylation is dispensable, in EGF-induced mitogenesis.

A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Flt tyrosine kinase receptor family (Flt-1 and KDR/Flk-1).

Hepatocyte Growth Factor (HGF)/Scatter Factor secreted from sinusoidal endothelial cells and Kupffer cells in liver activates the c-Met tyrosine kinase receptor expressed on hepatocytes. Here we report yet another possible communication system through a different ligand and tyrosine kinase receptor in an opposite direction. We isolated and determined the primary structure of the entire coding region of rat flt-1 (fms-like tyrosine kinase), a receptor for Vascular Endothelial Growth Factor (VEGF). Using rat flt-1 cDNA as a probe we found that the flt-1 mRNA was expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. The transcripts of another VEGF receptor KDR/Flk-1 structurally related to Flt-1 was also expressed specifically in sinusoidal endothelial cells. On the other hand, VEGF mRNA was expressed weakly in hepatocytes, but not in the nonparenchymal cell fraction. Furthermore, in an in vitro culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells. These results suggest that hepatocytes regulate the proliferation and survival of the sinusoidal endothelial cells in liver in a paracrine manner. Therefore two reciprocal communication systems, VEGF-Flt receptor family and HGF-Met receptor, may exist in hepatic tissue.

Rates of interactions of superoxide with vitamin E, vitamin C and related compounds as measured by chemiluminescence.

The rate constants for the interactions of superoxide with vitamin E (alpha-tocopherol), vitamin C (ascorbic acid) and their related compounds have been measured by a chemiluminescence method. A strong chemiluminescence of a constant intensity was observed when xanthine oxidase was added to an aqueous solution of hypoxanthine and a Cypridina luciferin analog, 2-methyl-6-phenyl-3-7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA). Vitamin E, vitamin C and their related compounds competed with CLA to react with superoxide and reduced the chemiluminescence intensity. From a kinetic analysis of the effect of addition of these compounds on the chemiluminescence intensity, the rate constants for their interactions with superoxide were measured at 25 degrees C and pH 7.8. The rate constants were obtained as 3.3 x 10(5) and 1.7 x 10(4) M-1 s-1 for ascorbate and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, respectively, and also as 4.9 x 10(3) and 4.5 x 10(3) M-1 s-1 for alpha-tocopherol incorporated into soybean and dimyristoyl phosphatidylcholine liposomal membranes, respectively. It has been shown that this method is a sensitive and a quick method which can be applied for measurement of the reactivities of various natural and synthetic compounds toward superoxide. In addition it has been shown that this method can also be applied to the heterogeneous system as well as homogeneous solution, which makes it more versatile and useful for the study in biochemistry.

Effects of ebselen and probucol on oxidative modifications of lipid and protein of low density lipoprotein induced by free radicals.

The oxidative modification of low density lipoprotein (LDL) is accepted to be an important early event of atherosclerosis, but it has not yet been well understood. The preventive effects of two antioxidants with different functions, ebselen and probucol, against the oxidative modification of LDL induced by copper or a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied in order to elucidate the mechanism of modification of apolipoprotein B-100 (apoB). Ebselen inhibited the copper-induced oxidation completely by reducing the hydroperoxides in LDL, since the initiation of copper-dependent oxidation requires the presence of a trace amount of hydroperoxides in LDL. On the other hand, ebselen did not suppress the oxidations of LDL induced by AAPH which generated free radicals by its thermal decomposition. The AAPH-induced oxidation of LDL in the absence of ebselen gave phosphatidylcholine hydroperoxide and cholesteryl ester hydroperoxide as major products, while in its presence, the hydroperoxides were reduced to corresponding alcohols. Interestingly, ebselen had little effect on the increase of relative electrophoretic mobility and fragmentation of intact apoB in the AAPH-induced oxidation. Probucol inhibited the oxidation of lipids in LDL effectively induced by either copper or AAPH, but the protein modifications were observed even in the presence of probucol. It was suggested that (1) lipid hydroperoxides do not play an important role in the modification of apoB such as increase in negative charge and fragmentation, (2) the direct attack of free radicals upon apoB and its modification by lipid oxidation products derived from hydroperoxides increase the negative charge of apoB, and (3) its fragmentation is caused primarily by an attack of free radicals.

Dynamics of the oxidation of low density lipoprotein induced by free radicals.

Although the oxidation of low density lipoprotein (LDL) has been studied extensively, its dynamics have received much less attention. The present study was carried out aiming to elucidate the rates and products of the oxidation of LDL induced by free radicals generated in three different ways by hydrophilic and lipophilic azo radical initiators and copper. The oxidations were followed by measuring the formation of lipid oxidation products, the uptake of oxygen, consumption of vitamin E, and the fragmentation and acquisition of negative charge by apolipoprotein B within a single oxidation reaction. LDL was oxidized by a free radical chain mechanism independent of the manner of initiating free radical generation to give phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH) as major primary products. When the radicals were generated within the LDL compartment, CEOOH was formed almost exclusively and quantitatively at the initial stage. The kinetic chain length for the oxidation of cholesteryl ester was larger than that for phosphatidylcholine in the presence and absence of vitamin E even when the radicals were generated initially in the aqueous phase. PCOOH and CEOOH accumulated with increasing extent of oxidation, but then decreased at the later stage of oxidation. The decrease in hydroperoxides was more significant in copper-induced oxidations. The thiobarbituric acid reactive substances also increased with time, but they accounted for less than 10% of total oxygen uptake. Oxidative modification of apo B was also observed. The modification of apo B, that is, fragmentation and increase in negative charge correlated well with the oxidation of lipids independent of the manner of chain initiation. It was also suggested that some radicals formed in the aqueous phase attacked apo B directly. These results suggest the importance of chain propagation in the oxidative modification of LDL and also the lipophilic, chain-breaking antioxidants within LDL in its inhibition.

Antioxidant activities of probucol against lipid peroxidations.

The antioxidant activities of probucol were measured in the oxidations of methyl linoleate in homogeneous solution and soybean phosphatidylcholine liposomal membranes and also of low-density lipoproteins. When an excess amount of probucol was reacted with galvinoxyl, the EPR spectrum of galvinoxyl disappeared and a new triplet EPR signal was found: g = 2.0058 and aH(2H) = 0.14 mT. The identical EPR spectrum was observed when probucol was reacted with tert-butoxyl radical generated from di-tert-butylperoxy oxalate. This EPR signal disappeared rapidly when reacted with either alpha-tocopherol or 6-O-palmitoyl-ascorbic acid. Probucol suppressed the free-radical-mediated oxidations of methyl linoleate in hexane and in acetonitrile, in a dose-dependent manner. Its antioxidant activity was 17.5-fold less than that of alpha-tocopherol in hexane. Probucol incorporated into soybean phosphatidylcholine liposomes suppressed its oxidation. The antioxidant activity of probucol was less than that of alpha-tocopherol, but the difference between the two antioxidant activities was smaller in the membranes than in homogeneous solution. Probucol also suppressed the oxidation of low-density lipoprotein. Interestingly, probucol suppressed the oxidation of LDL as efficiently as alpha-tocopherol, implying that physical factors as well as chemical reactivity are important in determining the overall activity of antioxidant in low-density lipoprotein.

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli.

The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG).

Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity.

We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity. Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis-Menten kinetics. A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and [3H]DFP covalently labeled protein D2. We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity. One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody. In addition, the fractions derived from the outer membrane of the protein D2-deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2-positive parent strain showed strong protease activity.