Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C-P bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 A X-ray crystal structure of the mutant Lys53Arg complexed with Mg2+ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain-core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH4 in the presence of Pald was determined at 2.4A resolution to reveal N epsilon-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C2 of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain N epsilon-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P-C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P-C bond cleavage.     

Asymmetric synthesis of chiral 2-hydroxy ketones by coupled biocatalytic alkene oxidation and CC bond formation

Two different biocatalytic reactions – a CC cleavage and a CC forming reaction – were evaluated concerning their application in a reaction sequence. In the overall reaction, an aromatic alkene was converted to a chiral 2-hydroxy ketone. In the first step, the olefin trans-anethole was converted to para-anisaldehyde and acetaldehyde by an aqueous extract of the white rot fungus Trametes hirsuta G FCC 047. The selective oxidative cleavage of the carbon–carbon double bond was achieved using molecular oxygen as a substrate. In a second step p-anisaldehyde was ligated to acetaldehyde to yield either (R)- or (S)-2-hydroxy-1-(4-methoxyphenyl)-propanone. The reaction was catalyzed by the enantiocomplementary CC bond forming enzymes benzaldehyde lyase and benzoylformate decarboxylase, respectively.     

Kinetic evidence for a substrate-induced fit in phosphonoacetaldehyde hydrolase catalysis

Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4-dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK(a) of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover.     

Half-sandwich Ru[9]aneS3 complexes structurally similar to antitumor-active organometallic piano-stool compounds: Preparation, structural characterization and in vitro cytotoxic activity

The preparation, structural characterization, and chemical behavior in aqueous solution of a series of new Ru[9]aneS₃ half-sandwich complexes of the type [Ru([9]aneS₃)Cl(NN)][CF₃SO₃] and [Ru([9]aneS₃)(dmso-S)(NN)][CF₃SO₃]₂ (5–15, NN = substituted bpy or 2 × 1-methylimidazole) are described. The X-ray structures of [Ru([9]aneS₃)Cl(3,3′-H₂dcbpy)][CF₃SO₃] (9) (3,3′-H₂dcbpy = 3,3′-dicarboxy-2,2′-bipyridine), [Ru([9]aneS₃)Cl(4,4′-dmobpy)][CF₃SO₃] (13) (4,4′-dmobpy = 4,4′-dimethoxy-2,2′-bipyridine), and [Ru([9]aneS₃)Cl(1-MeIm)₂][CF₃SO₃] (15) (1-MeIm = 1-methylimidazole) were also determined. The new compounds are structurally similar to anticancer-active organometallic half-sandwich complexes of formula [Ru(η⁶-arene)Cl(NN)][PF₆]. Three chloro compounds (5, 9, 15) were tested in vitro for cytotoxic activity against two human cancer cell lines in comparison with the previously described [Ru([9]aneS₃)Cl(en)][CF₃SO₃] (1, en = ethylenediamine), [Ru([9]aneS₃)Cl(bpy)][CF₃SO₃] (2), and with their common dmso precursor [Ru([9]aneS₃)Cl(dmso-S)₂][CF₃SO₃] (3). Only the ethylenediamine complex 1 showed some antiproliferative activity, ca. one order of magnitude lower than the reference organometallic half-sandwich compound RM175 that contains biphenyl instead of [9]aneS₃. This compound was further tested against a panel of human cancer cell lines (including one resistant to cisplatin).     

Kinetics of ammonium, nitrate and phosphorus uptake by Canna indica and Schoenoplectus validus

Canna indica L. is an upright perennial rhizomatous herb, and Schoenoplectus validus (Vahl) A. Löve and D. Löve is a tall, perennial, herbaceous sedge. The nutrient uptake kinetics of C. indica and S. validus were investigated using the modified depletion method after plants were grown for 4 weeks in simulated secondary-treated wastewater. The maximum uptake rate (I_max) and Michaelis–Menten constant (K_m) were estimated by iterative curve fitting. The I_max for NH₄N (623 μmol g⁻¹ dry root weight h⁻¹) was significantly higher than that for NO₃N (338 μmol g⁻¹ dry root weight h⁻¹) in S. validus. In contrast, no difference was observed in C. indica. The I_max values for NO₃N and NH₄N were higher in S. validus than in C. indica. A significantly lower K_m was detected for NO₃N uptake in C. indica (385 μmol L⁻¹) compared to that in S. validus (1908 μmol L⁻¹). The I_max for PO₄P did not differ between the plant species. The K_m for PO₄P was significantly higher in C. indica (157 μmol L⁻¹) than in S. validus (60 μmol L⁻¹). In conclusion, we found that S. validus preferred NH₄N over NO₃N, had greater capacity for N uptake and higher affinity for PO₄P, but C. indica had greater affinity for NO₃N. Nutrient uptake capacity is likely related to habitat preference, and is influenced by the structure of roots and rhizomes.     

X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Pald carbonyl. The x-ray crystal structures of the wild-type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 A, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Pald carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 epsilon-amino group, positioned for reaction with the Pald carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in k(cat)/K(m) upon Ala substitution) and Met-49 (17,000-fold reduction in k(cat)/K(m) upon Leu substitution) function in catalysis of Schiff-base formation. Based on these results, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met-49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Pald carbonyl and that substrate binding at the core domain stabilizes cap domain binding.     

Protein kinase C-α negatively regulates EGF-induced PLC- activity through direct phosphorylation

Phospholipase C- is a PLC isozyme that contains a CDC25 homology domain and a pair of RA domains in addition to a conserved PLC catalytic domain. PLC- is activated by both growth factors and GPCR ligands in a distinct manner. Growth factors such as EGF stimulate PLC- in an RA2 domain-dependent manner through Ras and Rap. On the other hand, several GPCR ligands that are linked with Ga₁₂ or Ga₁₃ can activate PLC- by associating with GTP-RhoA. GTP-RhoA binds with the region in the PLC- Y domain. Gs-linked ligands such as PGE1 and adrenaline stimulate PLC- by cAMP-dependent activation of Epac and Rap2B. PLC- is important for cardiac development and function. In addition, several lines of evidence indicate that PLC- promotes cell growth in an activity-dependent or -independent manner. In particular, PLC--dependent suppression of EGF receptor downregulation contributes to its growth promoting activity. Proper regulation of PLC- activity is essential for preventing tumor formation. Our previous report indicated that EGF-dependent ubiquitination of PLC- is required for the control of PLC--dependent cell growth. Recently, we found that PLC- is phosphorylated by growth factor stimulation, and this is another mechanism of the negative regulation. PLC- is phosphorylated by PKC-α upon stimulation with growth factors such as EGF and PDGF. The EGF-induced phosphorylation of PLC- was abolished by PKC inhibitors and by the expression of the dominant negative mutant of PKC-α. Furthermore, PKC-α was found to phosphorylate PLC- directly in vitro, suggesting that PLC- is a substrate of PKC-α in cells. In addition, PLC- was co-immunoprecipitated with PKC-α in an EGF-dependent manner. Immunocytochemical studies showed that PLC- co-localized with PKC-α in the plasma membrane after EGF stimulation. In addition, inhibition of PKC activity enhanced PLC--mediated PIP₂ hydrolysis, suggesting that PKC-α negatively regulates PLC- activity. Taken together, these results suggest for the first time that PLC- is regulated by PKC-α-dependent phosphorylation.     

CHMetal(II) axial interaction in planar complexes (metal = Cu, Pd) and implications for possible environmental effects of alkyl groups at biological copper sites

Intramolecular M(II)H–C interactions (M(II)=Cu(II), Pd(II)) involving a side chain alkyl group of planar d⁸ and d⁹ metal complexes of the N-alkyl (R) derivatives of N,N-bis(2-pyridylmethyl)amine with an N₃Cl donor set were established by structural and spectroscopic methods. The methyl group from the branched alkyl group (R = 2,2-dimethylpropyl and 2-methylbutyl) axially interacts with the metal ion with the MC and MH distances of 3.056(3)–3.352(9) and 2.317(1)–2.606(1) Å, respectively, and the M–H–C angles of 122.4–162.3°. The Cu(II) complexes showing the interaction have a higher redox potential as compared with those without it, and the ¹H NMR signals of the interacting methyl group in Pd(II) complexes shifted downfield relative to the ligand signals. Dependence of the downshift values on the dielectric constants of the solvents used indicated that the M(II)H–C interaction is mainly electrostatic in nature and may be regarded as a weak hydrogen bond. Implications for possible environmental effects of the leucine alkyl group at the type 1 Cu site of fungal laccase are also discussed.     

Biotransformation of sesquiterpenoids having α,β-unsaturated carbonyl groups with cultured plant cells of Marchantia polymorpha

The biotransformation of sesquiterpenoids having an α,β-unsaturated carbonyl group, such as α-santonin (1), lancerodiol p-hydroxybenzoate (2), 8,9-dehydronootkatone (3), and nootkatone (4), with cultured suspension cells of Marchantia polymorpha was investigated. It was found that the CC double bond of 1 and 2 was hydrogenated to give 1,2-dihydro-α-santonin (5) and 3,4-dihydrolancerodiol p-hydroxybenzoate (6), respectively, while the allylic position of the CC double bond of 3 and 4 was hydroxylated to give 13-hydroxy-8,9-dehydronootkatone (7) and 9-hydroxynootkatone (8), respectively.     

Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction

The determination of the redox properties of the cofactor in heme proteins provides fundamental insight into the chemical characteristics of this wide-spread class of metalloproteins. For the preparation of the ferroheme state, probably the most widely applied reductant is sodium dithionite, which at neutral pH has a reduction potential well below the reduction potential of most heme centers. In addition to the heme iron, some heme proteins, including the nitrophorins (NPs), contain cysteinecysteine disulfide bonds. In the present study, the effect of dithionite on the disulfides of NP4 and NP7 is addressed. To gain deeper understanding of the disulfide/dithionite reaction, oxidized glutathione (GSSG), as a model system, was incubated with dithionite and the products were characterized by ¹³C NMR spectroscopy and reverse phase chromatography in combination with mass spectrometry. This revealed the formation of one equivalent each of thiol (GSH) and glutathione-S-thiosulfate (GSSO₃⁻). With this background information, the effect of dithionite on the cystines of NP4 and NP7 was studied after trapping of the thiols with para-cloromercurybenzyl sulfonate (p-CMBS) and subsequent matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) where the heterolytic cleavage of the SS bond appears with only 2 molar equivalents of the reductant. Furthermore, prolonged electrochemical reduction of NP4 and NP7 in the presence of electrochemical mediators also leads to disulfide breakage. However, due to sterical shielding of the disulfide bridges in NP4 and NP7, the cystine reduction can be largely prevented by the use of stoichiometric amounts of reductant or limited electrochemical reduction. The described disulfide breakage during routine iron reduction is of importance for other heme proteins containing cystine(s).     

The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.     

Regulatory links between PLC enzymes and Ras superfamily GTPases: Signalling via PLC

A growing body of work implies that links between PLC isoforms, in particular PLC, and small G-proteins from Ras superfamily could be important in regulation of a number of cellular processes. Through successful use of biochemistry and structural biology, several interactions have been characterized providing some ideas about the regulatory mechanisms. A number of signalling pathways have also been suggested that could involve direct interaction of Ras and Rho GTPases with PLC. Importantly, several studies combining cell biology and genetics have provided new insights into functions of PLC and highlighted the importance of this approach to extend further and consolidate currently incomplete picture regarding its roles in development and disease.     

Purification and some properties of -poly-l-lysine-degrading enzyme from Kitasatospora sp. CCTCC M205012

An -poly-l-lysine-degrading enzyme (PLD) from Kitasatospora sp. CCTCC M205012 has been purified to homogeneity by three steps of anion-exchange chromatography including DEAE-Sepharose, Source 15Q and Mono Q, with a 500-fold increase in specific activity and 40.9% yield. The PLD has a molecular mass of approximately 87.0 kDa and consists of two identical subunits with a molecular mass of 43.6 kDa. Electrophoretic shows that the PLD isoelectric point was about 7.2. The optimum temperature and pH for the PLD was 30 °C and 7.0, respectively. The PLD was deactivated by EDTA, which was indicated that the enzyme was a metallo enzyme. The activity of PLD was stimulated by Co²⁺ and inhibited by Ca²⁺ remarkably. The apparent K_m with l-lysyl-p-nitroanilide as substrate was 0.216 mM and the V_max was 0.112 mmol/min mg. The PLD was an exo-type enzyme and monomers of l-lysine were detected during the enzymatic degradation of -PL.     

Fine structure characterization of amylopectins from grain amaranth starch

The aim of this study was to determine the fine structure of amylopectin from grain amaranth. Amaranthus amylopectin was hydrolyzed with α-amylase, and single clusters and a group of clusters (domain) were isolated by methanol precipitation. The domain and the clusters were treated with phosphorylase a and then β-amylase to remove all external chains, whereby the internal structure was obtained. The ,β-limit dextrins were analyzed on Sepharose CL 6B. The average DP (degree of polymerization) and peak-DP values of fractions of clusters were 57 and 82, respectively; the values of the domain were 137 and 309, respectively. The unit chain length profiles were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC–PAD). The results showed that the domain fraction contained 2.2 clusters, and single clusters were composed of 13 chains. The ,β-limit dextrins of the clusters were further hydrolyzed with α-amylase to characterize their building block composition. The average DP of the branched blocks was 11 and they contained on average 2.5 chains. Their average chain length, internal chain length, and degree of branching were approximately 4.3, 2.8, and 14, respectively. A cluster consisted of 6 branched blocks, and the internal chain length between the blocks was 6.8.     

Adult Brugia malayi 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha

We earlier reported a sizeable protection conferred by ‘mitochondria rich’ (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of  34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-α, IFN-γ and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that  34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.     

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Nitric oxide (NO) is thought to react with fatty acid alkoxyl radical, which is generated from fatty acid hydroperoxide via one-electron reduction. However, detail in the reaction remains obscure. In the present study, we examined the reaction of nitric oxide with fatty acid alkoxyl radical generated in the lipoxygenase/linoleate/13-hydroperoxyoctadecadienoate (13-HpODE) system under anaerobic conditions via HPLC equipped with mass spectrometry and photodiode array detections. In this reaction system, nitric oxide can scavenge linoleate alkoxyl radical, producing 13-ONO-9Z,11E-ODE. However, instead of 13-ONO-9Z,11E-ODE, 13-NO-9E,11E-ODE and 9-NO-10E,12E-ODE were alternatively detected in the reaction solution. To explain this contradiction, we proposed a mechanism as follows: (1) 13-ONO-9E/11Z-ODE undergoes homolytic cleavage at >CHONO bond into the linoleate allyl radical and nitrogen dioxide, (2) the allyl radical undergoes resonance stabilization into the E/E-form, and (3) nitric oxide scavenges the E/E-pentadiene radical at C9 or C13 position. Consequently, we concluded that nitric oxide immediately converts fatty acid alkoxyl radical into allyl radical.     

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.     

A review of the Cambrian biostratigraphy of South Australia

Cambrian rocks in South Australia occur in the Stansbury, Arrowie, eastern Officer and Warburton Basins. The succession in the Stansbury and Arrowie Basins can be divided into three sequence sets (supersequences), 1, 2 and 3. Sequence set 1 can be divided into five third-order sequences: 1.0, 1.1A, 1.1B, 1.2 and 1.3. Trilobites from the Stansbury and Arrowie Basins are restricted largely to the lower part of the succession. Four trilobite zones are recognized: Abadiella huoi (latest Atdabanian–earliest Botoman), Pararaia tatei, Pararaia bunyerooensis and Pararaia janeae Zones (all Botoman). Trilobites higher in the succession are known from only a few horizons and in part correlate with the upper Lower Cambrian Lungwangmiaoan Stage of China, equivalent to the top Toyonian. Pagetia sp. has been reported in the Coobowie Formation of the Stansbury Basin, thus suggesting an early Middle Cambrian age.The Cambrian faunas of the Warburton Basin range in age from early Middle Cambrian (Late Templetonian) to very Late Cambrian, although the richest faunal assemblages are late Middle Cambrian (Ptychagnostus punctuosus to Goniagnostus nathorsti Zones). Conodonts, including Cordylodus proavus, occur in a Datsonian fauna.The Arrowie Basin contains the most complete and best studied archaeocyath succession in the Australia–Antarctica region. The Warriootacyathus wilkawillensis, Spirillicyathus tenuis and Jugalicyathus tardus Zones from the lower Wilkawillina Limestone (Arrowie Basin) and equivalents are correlated with the Atdabanian. Botoman archaeocyathids occur higher in the Wilkawillina Limestone. The youngest (Toyonian) archaeocyath fauna in Australia occurs in the Wirrealpa Limestone (Arrowie Basin).Brachiopods and molluscs of the Arrowie and Stansbury Basins can be divided into four biostratigraphic assemblages. Several informal Early Cambrian SSF biostratigraphic assemblages are recognized. Probable tabulate-like corals occur in the Botoman Moorowie Formation. Seven informal acritarch assemblages occur in the Early Cambrian of the Stansbury and Arrowie Basins. Trace fossils may mark the Precambrian–Cambrian boundary. Only two of several tuffaceous horizons from the Stansbury and Arrowie Basins have been dated (i) a date of 522.0 ± 2.1 Ma from the Heatherdale Shale of the Stansbury Basin, about 400 m above latest Atdabanian archaeocyathids and (ii) a date of 522.0 ± 1.8 Ma from the lower part of the Billy Creek Formation in the Arrowie Basin. Neither date is regarded as reliable.