Risk factors for women with gestational diabetes mellitus developing type 2 diabetes and the impact on children's health.

AIM: The study aimed to explore the risk factors for progression to type 2 diabetes in women with gestational diabetes mellitus and the long-term impact of gestational diabetes mellitus on the health of their children. BACKGROUND: The occurrence of gestational diabetes mellitus in women is the strongest predictor of the development of type 2 diabetes in the future. DESIGN: A non-concurrent cohort and case-control study design and the guidelines for STROBE checklist were used for the study. METHODS: We reviewed medical records for the time period from the archives of the hospitals. The short-listed women and their children were invited to participate in a 'Women with gestational diabetes mellitus' research activity from November 2012-February 2016. Blood tests, physiological indicators and demographic questionnaires were applied for data collection. RESULTS: A total of 57 women and 57 children participated in the research activity. The risk factors for progression of gestational diabetes mellitus to type 2 diabetes included education level and the 1-h blood glucose level for the antepartum 100-g oral glucose tolerance test and the 2-h blood glucose level for the postpartum 75-g oral glucose tolerance test. However, these risk factors did not have a significant impact on the body mass index and body fat of the women's children. CONCLUSION: Healthcare providers should adopt a breadth of health education strategies to remind women with gestational diabetes mellitus to be aware of certain risk factors, and to urge them and their children to return for regular follow-up visits. RELEVANCE TO CLINICAL PRACTICE: Identification of the risk factors developing type 2 diabetes can make healthcare units incorporate the postpartum follow-up of gestational diabetes mellitus into their standard protocol and case managers continue to follow-up and manage the health of the women and that of their children to reduce adverse health of them.

The Impact of Prepregnancy Body Mass Index and Gestational Weight Gain on Perinatal Outcomes for Women With Gestational Diabetes Mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM), an obstetric disease that affects the health of pregnant women, is one of the key factors associated with perinatal mortality or disease. AIMS: To explore the impact of prepregnancy body mass index (BMI) and gestational weight gain (GWG) on perinatal outcomes for women with GDM. METHODS: With a retrospective study design, women were recruited who received prenatal checkups, gave birth at two teaching hospitals in southern Taiwan from 1995 to 2011, and received a diagnosis of GDM by an obstetrician. A trained research assistant collected the participants' data in each hospital's archives room. The researcher used a retrospective case study method to identify women who received a GDM diagnosis between 1995 and 2011. RESULTS: Women with GDM and with an overweight prepregnancy BMI were more likely to have cesarean deliveries and to use glucose-lowering medicines after delivery. Their newborns also had a higher birth weight. In addition, gestational hypertension and cesarean delivery were more common in women with GDM and with excessive GWG than in women with GDM and with normal GWG. The newborns of women with GDM and with excessive GWG had higher birth weights and more nuchal cord than those of women with GDM and with normal GWG. More women with GDM and with excessive GWG underwent blood glucose monitoring than did women with GDM and with normal GWG. LINKING EVIDENCE TO ACTION: The prepregnancy weight and GWG significantly affected perinatal outcomes in both the women with GDM themselves and their newborns. Healthcare professionals must provide childbearing women with additional health education in the areas of health promotion, nutrition, weight control, exercise, and maintaining regular everyday lives.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

Fenofibrate suppresses melanogenesis in B16-F10 melanoma cells via activation of the p38 mitogen-activated protein kinase pathway.

Fenofibrate and ciglitazone belong to the classes of fibrates and thiazolidinediones, respectively. Their pharmacological actions on peroxisome proliferator-activated receptors (PPARs) present a potential therapy for hyperlipidemia and hyperglycemia. However, the melanogenesis affected by PPAR ligands in melanocytes has not been well investigated. By determining the melanin content of cells treated with PPAR agonists, we showed that fenofibrate significantly reduced melanin synthesis, but its major active metabolite, fenofibric acid, did not. Notably, the suppression of melanogenesis by fenofibrate could not be prevented by the PPARα specific antagonist GW6471. In addition, T0901317, a liver X receptor (LXR) agonist, restored the antimelanogenic activity of fenofibrate. Accordingly, fenofibrate may suppress melanogenesis through a PPARα-independent pathway. Treatment of cells with fenofibrate led to the down-regulated gene expression of melanocortin 1 receptor (MC1R). Fenofibrate also attenuated the dihydroxyphenylalanine (DOPA)-staining activity and expression of tyrosinase as well as the expression of microphthalmia-associated transcription factor (MITF). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was stimulated by fenofibrate. Furthermore, the p38 MAPK inhibitor SB203580 prevented the repressive effects of fenofibrate on the melanin production. Taken together, the results of the present study suggest that fenofibrate inhibits melanin synthesis via the down-regulation of MC1R, the up-regulation of p38 MAPK, and interference with LXR signaling pathways to decrease the expression of tyrosinase in B16-F10 melanoma cells.

Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2.

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.

Melanogenesis of murine melanoma cells induced by hesperetin, a Citrus hydrolysate-derived flavonoid.

Melanogenesis is a complex process that modulates skin pigmentation to defend photodamage. Citrus is the most widely produced fruit crop in the world. People ingest various citrus fruits in their common diets. In the present study, the acid-hydrolyzed and un-hydrolyzed extracts of orange-type citrus fruits were subjected to analyze flavonoid compositions and assess their effects on melanin synthesis in murine B16-F10 melanoma cells. The acid-hydrolyzed extracts of Citrus sinensis, C. reticulata, and C. aurantium enhanced melanin production. Based on high-performance liquid chromatography (HPLC) analysis, the most abundant flavonoids that were found in citrus hydrolyzed extracts were hesperetin and naringenin. Hesperetin exhibited the most potent activity on melanin synthesis and induced tyrosinase and microphthalmia-associated transcription factor (MITF) expression. Moreover, hesperetin stimulated the activation of mitogen-activated protein kinases (MAPKs), phosphorylation of cAMP-responsive element binding protein (CREB) and glycogen synthase kinase-3ß (GSK3ß), and subsequently induced the accumulation of ß-catenin. This study suggests that the citrus constituent hesperetin might have protective melanogenic potential as a cosmeceutical agent against skin photodamage.

Guanidination of notexin promotes its phospholipase A(2) activity-independent fusogenicity on vesicles with lipid-supplied negative curvature.

To address the requirement of phospholipase A(2) (PLA(2)) activity in membrane fusion events and membrane perturbation activity of notexin and guanidinated notexin (Gu-notexin), the present study was conducted. Notexin and Gu-notexin did not show PLA(2) activity after the removal of Ca(2+) with EDTA. Metal-free notexin and Gu-notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA(2) activity. Compared with Ca(2+)-bound Gu-notexin, fusogenicity of metal-free Gu-notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of notexin regardless of the absence or presence of Ca(2+). Consistently, metal-free Gu-notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free notexin, and fusion activity of metal-free Gu-notexin on cholesterol-depleted RBCs notably reduced. Compared with notexin, Gu-notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that notexin and Gu-notexin could induce vesicle leakage and fusion via a PLA(2) activity-independent mechanism, and guanidination promotes PLA(2) activity-independent fusogenicity of notexin on vesicles with lipid-supplied negative curvature.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.

Citrus flavanone naringenin enhances melanogenesis through the activation of Wnt/ß-catenin signalling in mouse melanoma cells.

Citrus fruits are the major source of flavonoids for humans, and flavanones are the main flavonoids in the Citrus species. Among the Citrus flavanones, the glycoside derivatives of naringenin, naringin and narirutin, are the most abundant in grapefruit. The present study aimed to investigate the molecular events of melanogenesis induced by naringenin in murine B16-F10 melanoma cells. Melanin content, tyrosinase activity and Western blot analysis were performed to elucidate the possible underlying mechanisms. Exposure of melanoma cells to naringenin resulted in morphological changes accompanied by the induction of melanocyte differentiation-related markers, such as melanin synthesis, tyrosinase activity, and the expression of tyrosinase and microphthalmia-associated transcription factor (MITF). We also observed an increase in the intracellular accumulation of ß-catenin as well as the phosphorylation of glycogen synthase kinase-3ß (GSK3ß) protein after treatment with naringenin. Moreover, the activity of phosphatidylinositol 3-kinase (PI3K) was up-regulated by naringenin since the phosphorylated level of downstream Akt protein was enhanced. Based on these results, we concluded that naringenin induced melanogenesis through the Wnt-ß-catenin-signalling pathway.

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2.

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A(2) (PLA(2)). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA(2) toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA(2). Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA(2). Moreover, it was found that PLA(2) and N-terminally mutated PLA(2) adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA(2) and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA(2), our data indicate that sphingomyelin modulates the mode of membrane binding of PLA(2) at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA(2).

Lipid domain formation modulates activities of snake venom phospholipase A(2) enzymes.

The goal of the present study is to elucidate the effect of lipid domain formation on activities of Naja naja atra and Bungarus multicinctus phospholipase A(2) (PLA(2)) enzymes. Sphingomyelin inhibited enzymatic activity and membrane-damaging activity of PLA(2) against egg yolk phosphatidylcholine (EYPC), while cholesterol and cholesterol sulfate abrogated the inhibitory effect of sphingomyelin. The ability of cholesterol and cholesterol sulfate to abolish the inhibitory effect of sphingomyelin was closely related to their capacity to induce domain formation in EYPC/sphingomyelin vesicles. Laurdan fluorescence measurement revealed that membrane packing of EYPC/sphingomyelin vesicles was differently affected by cholesterol and cholesterol sulfate. Unlike cholesterol, cholesterol sulfate was unable to promote domain formation in dipalmitoylphosphatidylcholine (DPPC) vesicles. Cholesterol increased but cholesterol sulfate reduced PLA(2) activity against DPPC. Self-quenching studies and trinitrophenylation of Lys residues revealed that PLA(2) enzymes adopted different membrane-bound mode upon absorption onto the membrane bilayers comprised of different lipid compositions. Collectively, our data indicate that lipid domain formation regulates PLA(2) activity, and suggest that the physical state of membrane bilayers changes the interactive mode of PLA(2) with phospholipids.

Taiwan cobra phospholipase A2 elicits posttranscriptional up-regulation of ADAM17 in human neuroblastoma SK-N-SH cells.

Taiwan cobra phospholipase A(2) (PLA(2)) treatment promoted proADAM17 processing into mature ADAM17 in human neuroblastoma SK-N-SH cells. The abolishment of catalytic activity caused a drastic drop in the PLA(2) ability to induce ADAM17 maturation, and lysophosphatidylcholine treatment mimicked the effect of PLA(2). ADAM17 activity measurement, ADAM17 cell surface levels, TNFR2 ectodomain shedding, and ADAM17 mRNA transcription supported that posttranscriptional up-regulation of ADAM17 occurred in PLA(2)-treated SK-N-SH cells. PLA(2) treatment induced p38 MAPK activation and ERK inactivation. p38 MAPK activation suppression by SB202190 (p38 MAPK inhibitor) abolished posttranscriptional up-regulation of ADAM17 in PLA(2)-treated cells, while treatment with U0126 (MEK1 and MEK2 inhibitor) increased ADAM17 maturation in SK-N-SH cells. Constitutively active MEK1 expression abrogated PLA(2)-induced ADAM17 maturation. Taken together, our data indicate that PLA(2)-evoked p38 MAPK activation and ERK inactivation are involved in ADAM17 posttranscriptional up-regulation, and suggest that the action of PLA(2) is catalytic activity-dependent.

Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol.

To elucidate the contribution of phospholipase A 2 (PLA2) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA 2 activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca2+-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA 2 activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.

Roles of lysine residues and N-terminal alpha-amino group in membrane-damaging activity of Taiwan cobra cardiotoxin 3 toward anionic and zwitterionic phospholipid vesicles.

In contrast to a slight increase in activity toward phosphatidylcholine (EYPC)/dimyristoyl phosphatidic acid (DMPA) vesicles, guanidination of Naja naja atra cardiotoxin 3 (CTX3) and selective trinitrophenylation of N-terminal alpha-amino group enhanced notably membrane-damaging activity on EYPC/egg yolk sphingomyelin (EYSM) vesicles. Chemically modified CTX3 showed a reduction in its hemolytic activity and cytotoxicity. These reflected that membrane-damaging activity of CTX3 was affected by phospholipid compositions. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles did not closely correlate with membrane-damaging potency of native and modified CTX3. Moreover, different topographical contacts and distinctive modes for the binding of CTX3 and its modified derivatives with anionic phospholipid vesicles (EYPC/DMPA) and zwitterionic phospholipid vesicles (EYPC/EYSM) were observed. Compared with in the case of EYPC/DMPA, the interaction between CTX molecules and EYPC/EYSM was drastically reduced by increasing salt concentration and heparin. Taken together, our data indicate that guanidination of Lys residues and trinitrophenylation of alpha-amino group alter differently the interacted modes upon absorption on anionic phospholipid vesicles and zwitterionic phospholipid vesicles. The findings also suggest that positively charged residues of CTX3 play a distinctive role in damaging anionic and zwitterionic phospholipid vesicles.

JNK1/c-Jun and p38 alpha MAPK/ATF-2 pathways are responsible for upregulation of Fas/FasL in human chronic myeloid leukemia K562 cells upon exposure to Taiwan cobra phospholipase A2.

Fas and FasL expression upregulation was found in human leukemia K562 cells upon exposure to Naja naja atra phospholipase A(2) (PLA(2)). PLA(2) treatment induced an increase in intracellular Ca(2+) ([Ca(2+)]i) and ROS generation levels, leading to activation of p38 MAPK and JNK. Suppression of both p38 MAPK and JNK abrogated Fas and FasL upregulation. Unlike PLA(2), catalytically inactive PLA(2) treatment did not markedly increase Fas and FasL protein expression, and p38 MAPK activation was exclusively responsible for catalytically inactive PLA(2)-induced increase in Fas and FasL protein expression. Knockdown of p38 alpha MAPK and JNK1 by siRNA proved that p38 alpha MAPK and JNK1 were involved in ATF-2 and c-Jun phosphorylation, respectively. Compared with the p38 alpha MAPK/ATF-2 pathway, the JNK1/c-Jun pathway played a crucial role in Fas/FasL upregulation. Unlike arachidonic acid, lysophosphatidylcholine mimicked the PLA(2) action in inducing Fas/FasL upregulation. Together with the previous finding that c-Jun and ATF-2 are involved in transcriptional regulation of Fas and FasL, our data suggest that PLA(2) induces Fas and FasL upregulation through p38 alpha MAPK/ATF-2 and JNK1/c-Jun pathways in K562 cells, and PLA(2) catalytic activity is involved in this action.

Effect of cholesterol on membrane-damaging activity of Naja nigricollis toxin gamma toward phospholipid vesicles.

Membrane-damaging activity of Naja nigricollis toxin gamma on phospholipid vesicles was attenuated by incorporation of cholesterol into phospholipid vesicles. Deprivation of cholesterol from erythrocyte membrane enhanced notably hemolytic activity of toxin gamma, while the hemolytic activity of toxin gamma on cholesterol-depleted erythrocytes decreased when cholesterol was restored to membrane. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles were modestly affected in the presence of cholesterol. Time-resolved fluorescence and Fourier transform infrared spectra showed that phospholipid-bound toxin gamma and cholesterol/phospholipid-bound toxin gamma did not adopt the same conformation. Moreover, geometrical arrangement of toxin gamma in contact with phospholipid vesicles was different from that with cholesterol/phospholipid vesicles as evidenced by N-(fluorescein-5-thiocarbamoyl)-1, 2-dihexadecanoyl-phosphatidylethanolamine fluorescence enhancement and color transformation of phospholipid/polydiacetylene membrane assay. Taken together, our data show that ordered phospholipid phase arising from incorporation of cholesterol affects conformation and topographical arrangement of toxin gamma on water-lipid interface, thus attenuating its membrane-damaging activity against phospholipid vesicles.

Catalytic activity-independent pathway is involved in phospholipase A(2)-induced apoptotic death of human leukemia U937 cells via Ca(2+)-mediated p38 MAPK activation and mitochondrial depolarization.

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A(2) (PLA(2)), the present study is conducted to explore whether PLA(2) induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA(2)) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA(2). It was found that BPB-PLA(2)-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB-PLA(2), elevation of intracellular Ca(2+) levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca(2+) chelator) and nifedipine (L-type Ca(2+) channel blocker) abrogated Ca(2+) increase and p38 MAPK activation, and rescued viability of BPB-PLA(2)-treated U937 cells. BPB-PLA(2)-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA(2) mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA(2) activity, they induced death of U937 cells. The signaling pathway of PLA(2) mutants in inducing cell death was indistinguishable from that of BPB-PLA(2). Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA(2)-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca(2+)-mediated p38 MAPK activation.

Mutations on N-terminal region of Taiwan cobra phospholipase A(2) result in structurally distorted effects.

In the present study, three Taiwan cobra PLA(2) variants were prepared by adding an extra N-terminal Met, substituting Asn-1 by Met or deleting the N-terminal heptapeptide. Recombinant PLA(2) mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N-terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N-terminal heptapeptide or with an extra N-terminal Met added as evidenced by the inability of the two variants to bind with Ba(2+). Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca(2+)-induced change in the global structure of PLA(2) was different from that in N-terminal variants. Together with the fact that an 'activation network' connects the N-terminus with the active site, our data suggest that mutagenesis on the N-terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA(2).

Taiwan cobra cardiotoxins induce apoptotic death of human neuroblastoma SK-N-SH cells mediated by reactive oxygen species generation and mitochondrial depolarization.

Although Naja naja atra cardiotoxin 3 (CTX3) and cardiotoxin 4 (CTX4) showed different cytotoxicity toward human neuroblastoma SK-N-SH cells, the two toxins induced apoptotic death on SK-N-SH cells. The apoptosis signals of CTX3 and CTX3 included ROS generation, increase in mitochondrial permeability transition, cytochrome c release to the cytosol and activation of caspase-9 and -3. However, CTX3 quickly induced the effects with higher magnitude compared with CTX4. ROS production and subsequent apoptotic cell death in CTX-treated cells were partly blocked by the antioxidant 2,3-dihydroxybenzoic acid. Nevertheless, mitochondria alteration and cytosolic cytochrome c release were not significantly attenuated by the antioxidant. Cell death was not completely inhibited by caspase-3 inhibitor. Moreover, cyclosporine A, an inhibitor of mitochondrial permeability transition, slightly decreased CTX-induced ROS generation by approximately 15%. Taken together, our data indicate that N. naja atra CTXs induce ROS generation that is not wholly dependent on mitochondrial dysfunction, and that the cytotoxic potency of CTX3 and CTX4 on SK-N-SH cells is, at least in part, correlated with their capability in inducing ROS generation and mitochondrial alterations.

Mutations on the N-terminal region abolish differentially the enzymatic activity, membrane-damaging activity and cytotoxicity of Taiwan cobra phospholipase A2.

To examine the functional contribution of the N-terminal region to the activities of Naja naja atra phospholipase A(2) (PLA(2)), studies on three N-terminally mutated PLA(2) were carried out in the present work. Removal of N-terminal heptapeptide caused a complete loss of membrane-damaging activity, whilst the mutants with an extra Met before Asn-1 or substituting Asn-1 with Met still retained approximately 40.9% and 82.9% membrane-damaging activity of the native enzyme, respectively. Mutations on the N-terminal region did not greatly affect the Ca(2+)-binding ability but caused a precipitous drop in PLA(2) activity. Moreover, the gross conformation of the mutants was different from that of the native enzyme as revealed by CD spectra. Nevertheless, the mutants as well as native PLA(2) induced apoptotic death of U937 cells, and the cytotoxicity of mutants was similar to or even greater than that of the native PLA(2). These results indicate that mutations on the N-terminus abolish the enzymatic activity, membrane-damaging activity and cytotoxicity of N. naja atra PLA(2) in different ways, and suggest a feasible approach to selective elimination of the multiple activities of PLA(2) enzymes.