铜、铅、镉、锌、汞和银离子复合污染对水螅的急性毒性效应

以水螅(Hydrasp)为例,通过单因子静态急性毒性试验方法和等毒性溶液法,分别研究Hg2 、Cu2 、Cd2 、Ag 、Zn2 和Pb2 对其单一和复合毒性效应。单一实验结果表明,它们对水螅毒性大小顺序为Hg2 >Cu2 >Cd2 >Ag >Zn2 >Pb2 。复合毒性实验表明,Zn2 与Cu2 、Hg2 、Pb2 、Ag ;Pb2 与Cu2 ;Hg2 与Ag ;Pb2 与Ag 这些组合对水螅联合急性毒性总体上表现出拮抗作用,Cd2 与Cu2 、Hg2 、Pb2 、Ag 组合总体上则是协同作用,Zn2 与Cd2 、Pb2与Hg2 、Cu2 与Hg2 ,Ag 在不同的浓度水平组合下明显表现出不同的毒性效应。     

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.     

N2O as a substrate and as a competitive inhibitor of nitrogenase

We have investigated the inhibitory effect of N2O on NH3 formation by purified component proteins from Klebsiella pneumoniae and have confirmed that the inhibition is competitive with respect to N2 and that N2O is reduced to N2, which in turn is further reduced to NH3. In addition, we have shown that N2O is unable to support HD formation from D2 and H2O. N2-supported HD formation from D2 and H2O was found to be inhibited by N2O. In contrast to N2, N2O was found to suppress nitrogenase-mediated H2 evolution completely at infinitely high pN2O. H2 was found to inhibit N2O-supported NH3 production but not N2O-supported N2 production. The steady-state kinetics of N2O reduction showed a good fit to Michaelis-Menten kinetics with a Km for N2O of 5 mM at 30 degrees C, corresponding to 24 kPa of N2O. A model is proposed that fits the observed results.     

重金属对油菜种子萌发和胚根生长的影响

分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。     

Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.     

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.     

Differential binding to and regulation of JAK2 by the SH2 domain and N-terminal region of SH2-bbeta

SH2-Bbeta has been shown to bind via its SH2 (Src homology 2) domain to tyrosyl-phosphorylated JAK2 and strongly activate JAK2. In this study, we demonstrate the existence of an additional binding site(s) for JAK2 within the N-terminal region of SH2-Bbeta (amino acids 1 to 555) and the ability of this region of SH2-B to inhibit JAK2. Four lines of evidence support the existence of this additional binding site(s). In a glutathione S-transferase pull-down assay, wild-type SH2-Bbeta and SH2-Bbeta(R555E) with a defective SH2 domain bind to both tyrosyl-phosphorylated JAK2 from growth hormone (GH)-treated cells and non-tyrosyl-phosphorylated JAK2 from control cells, whereas the SH2 domain of SH2-Bbeta binds only to tyrosyl-phosphorylated JAK2 from GH-treated cells. Similarly, JAK2 is present in alphaSH2-B immunoprecipitates in the absence and presence of GH, with GH substantially increasing the coprecipitation of JAK2 with SH2-B. When coexpressed in COS cells, SH2-Bbeta coimmunoprecipitates not only wild-type, tyrosyl-phosphorylated JAK2 but also kinase-inactive, non-tyrosyl-phosphorylated JAK2(K882E), although to a lesser extent. DeltaC555 (amino acids 1 to 555 of SH2-Bbeta) that lacks most of the SH2 domain binds similarly to wild-type JAK2 and kinase-inactive JAK2(K882E). Experiments using a series of N- and C-terminally truncated SH2-Bbeta constructs indicate that the pleckstrin homology (PH) domain (amino acids 269 to 410) and amino acids 410 to 555 are necessary for maximal binding of SH2-Bbeta to inactive JAK2, but neither region alone is sufficient for maximal binding. The SH2 domain of SH2-Bbeta is necessary and sufficient for the stimulatory effect of SH2-Bbeta on JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B. In contrast, DeltaC555 lacking the SH2 domain, and to a lesser extent the PH domain alone, inhibits JAK2. DeltaC555 also blocks JAK2-mediated tyrosyl phosphorylation of Stat5B in COS cells and GH-stimulated nuclear accumulation of Stat5B in 3T3-F442A cells. These data indicate that in addition to the SH2 domain, SH2-Bbeta has one or more lower-affinity binding sites for JAK2 within amino acids 269 to 555. The interaction via this site(s) in SH2-B with inactive JAK2 seems likely to increase the local concentration of SH2-Bbeta around JAK2, thereby facilitating binding of the SH2 domain to ligand-activated JAK2. This would result in a more rapid and robust cellular response to hormones and cytokines that activate JAK2. This interaction between inactive JAK2 and SH2-B may also help prevent abnormal activation of JAK2.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.     

山东10种植物的核型分析

对山东１０ 种植物进行了核型分析。茴茴蒜（ Ｒａｎｕｎｃｕｌｕｓｃｈｉｎｅｎｓｉｓ Ｂｇｅ－） 染色体数目２ｎ ＝１６ , 核型公式Ｋ（２ｎ） ＝ ２ｘ ＝ １６ ＝ ２ Ｍ ＋ ２ｍ ＋ ２ｓｍ ＋ １０ｓｔ, “３Ａ”类型; 五脉地椒（ Ｔｈｙｍｕｓｑｕｉｎｑｕｅｃｏｓｔａｔｕｓ Ｃｅｌａｋ－） 染色体数目２ｎ＝ ２６ , 核型公式Ｋ （２ｎ） ＝ ２ｘ＝ ２６ ＝ ８ Ｍ ＋ １８ｍ , “１Ａ”类型; 蛇床（ Ｃｎｉｄｉｕｍ ｍｏｎｎｉｅｒｉ（Ｌ－） Ｃｕｓｓ－） 染色体数目２ｎ＝ ２０ , 核型公式Ｋ （２ｎ） ＝ ２ｘ＝ ２０ ＝ ２Ｍ＋ １６ｍ ＋ ２ｓｍ , “２Ｂ”类型; 波斯菊（ Ｃｏｓｍｏｓ ｂｉｐｉｎｎａｔｕｓ Ｃａｖ－） 染色体数目２ｎ ＝ ２４ , 核型公式Ｋ（２ｎ） ＝ ２ｘ ＝ ２４ ＝ １６ｍ ＋ ２ｍ （ｓａｔ） ＋ ６ｓｍ , “２Ａ”类型; 白车轴草（ Ｔｒｉｆｏｌｉｕｍ ｒｅｐｅｎｓ Ｌ－） 染色体数目２ｎ＝ ３２ , 核型公式Ｋ （２ｎ） ＝ ４ｘ ＝ ３２ ＝ ３２ｍ , “１Ａ”类型; 铁苋菜（ Ａｃａｌｙｐｈａ ａｕｓｔｒａｌｉｓ Ｌ－）染色体数目２ｎ ＝ ３２ , 核型公式Ｋ （２ｎ） ＝ ２ｘ＝ ３２ ＝ ３２ｍ , “１Ｂ”类型; 地构叶（ Ｓｐｅｒａｎｓｋｉａ ｔ?     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF

The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the functionality of the lipase in vivo. We demonstrate that stable short hairpin RNA (shRNA)-based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2(SiL)) into the Grb2-knockdown cells (COS7(shGrb2)), resulted in the nearly full rescue of PLD2 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7(shGrb2) cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespective of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments with Grb2 and PLD2-Y169/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGF. These results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo.     

MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase

MEK kinase 2 (MEKK2) is a 70-kDa protein serine/threonine kinase that has been shown to function as a mitogen-activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase domain in the COOH-terminal moiety of the protein. The NH(2)-terminal moiety of MEKK2 has no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2) was found to bind MEKK2; PRK2 has been previously shown to bind RhoA and the Src homology 3 domain of Nck. PRK2 did not bind MEKK3, which is closely related to MEKK2. The MEKK2 binding site maps to amino acids 637-660 in PRK2, which is distinct from the binding sites for RhoA and Nck. This sequence is divergent in the closely related kinase PRK1, which did not bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation of PRK2 is independent of MEKK2 regulation of the c-Jun NH(2)-terminal kinase pathway. MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK pathways and PRK2 regulated responses.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.     

2-MeOE2bisMATE induces caspase-dependent apoptosis in CAL51 breast cancer cells and overcomes resistance to TRAIL via cooperative activation of caspases

2-Methoxyoestradiol (2-MeOE2) is an endogenous oestrogen metabolite which inhibits tubulin polymerisation and has anti-tumour and anti-angiogenic activity. 2-MeOE2 induces apoptosis in a wide range of cancer cell types and has recently been demonstrated to cooperate with TRAIL to induce apoptosis in breast cancer cells. 2-Methoxyoestradiol-3,17-bis-O,O-sulphamate (2-MeOE2bisMATE) is a sulfamoylated derivative of 2-MeOE2 with enhanced activity and improved pharmacokinetic properties, and 2-MeOE2bisMATE is a promising candidate for early clinical trials. It is important, therefore, to understand the mechanisms by which 2-MeOE2bisMATE acts, and whether it retains the ability to cooperate with TRAIL. We demonstrate that 2-MeOE2bisMATE-induced apoptosis of CAL51 breast cancer cells was associated with rapid activation of caspase 3 and 9, but not caspase 8 (as measured by BID cleavage) and was completely prevented by the caspase inhibitor zVADfmk. Interfering with Fas- or TRAIL-receptor function did not prevent 2-MeOE2bisMATE-induced apoptosis. Whereas CAL51 cells were resistant to TRAIL-induced apoptosis, 2-MeOE2bisMATE and TRAIL cooperated to induce cell death. This apoptosis was associated with enhanced activation of caspases, but not increased expression of the DR5 TRAIL receptor, previously demonstrated to be induced by 2-MeOE2. Therefore, 2-MeOE2bisMATE-induced apoptosis is dependent on caspases and like 2-MeOE2, 2-MeOE2bisMATE can overcome resistance to TRAIL by stimulating activation of downstream caspases. Our results suggest that 2-MeOE2bisMATE and TRAIL might be a particularly effective combination of anti-cancer agents.