Characterization and expression of the maize ß-carbonic anhydrase gene repeat regions.

In maize, carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the first reaction of the C(4) photosynthetic pathway; it catalyzes the hydration of CO(2) to bicarbonate and provides an inorganic carbon source for the primary carboxylation reaction catalyzed by phosphoenolpyruvate (PEP) carboxylase. The ß-CA isozymes from maize, as well as other agronomically important NADP-malic enzyme (NADP-ME) type C(4) crops, have remained relatively uncharacterized but differ significantly from the ß-CAs of other C(4) monocot species primarily due to transcript length and the presence of repeat sequences. This research confirmed earlier findings of repeat sequences in maize CA transcripts, and demonstrated that the gene encoding these transcripts is also composed of repeat sequences. One of the maize CA genes was sequenced and found to encode two domains, with distinct groups of exons corresponding to the repeat regions of the transcript. We have also shown that expression of a single repeat region of the CA transcript produced active enzyme that associated as a dimer and was composed primarily of α-helices, consistent with that observed for other plant CAs. As the presence of repeat regions in the CA gene is unique to NADP-ME type C(4) monocot species, the implications of these findings in the context of the evolution of the location and function of this C(4) pathway enzyme are strongly suggestive of CA gene duplication resulting in an evolutionary advantage and a higher photosynthetic efficiency.

Synthesis of bifunctional saxitoxin analogues by biotinylation.

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety.

Biochemical and molecular characterisation of cubozoan protein toxins.

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as alpha-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.

Seroreactivity against streptococcal DRS (distantly related to SIC) protein is a predictor for end-stage renal failure.

We hypothesized that immunoreactivity against antigens from nephritic strains of Streptococcus pyogenes may be elevated in patients with end-stage renal failure (ESRF). Additionally, we investigated whether a difference in seroreactivity exists between nonindigenous and indigenous (Aboriginal/Torres Strait Islander) patients. To examine these possibilities, antibodies against potentially nephritogenic proteins, streptokinase (Ska1) (from M1), streptococcal pyrogenic exotoxin type B (SpeB) (from M1), the streptococcal inhibitor of complement-mediated cell lysis (SIC) (from M1) and its two variants, closely related to SIC (CRS) (from M57) and distantly related to SIC (DRS) (from M12) were determined in 66 patients and 31 healthy controls by enzyme-linked immunosorbent assays. A significantly higher proportion of patients compared to controls were seropositive to Ska1 (P = 0.004), DRS (P = 0.0003), CRS (P = 0.001), and SIC (P = 0.018). Regression analysis showed that seroreactivity to DRS (r(2) = 0.85, P = 0.001) predicted the development of ESRF and that being diabetic was positively associated with being an ESRF patient (r(2) = 0.37, P < 0.0001) and being indigenous (r(2) = 0.47, P < 0.0001). These results suggest that these ESRF patients were exposed to strains of S. pyogenes that secrete Ska1, DRS, CRS, and SIC and may have pathological significance. No significant difference was observed between the indigenous patients and nonindigenous patients.

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri.

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri.

Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal delta-endotoxins Cry1Aa, Cry3Bb and Cry3A.

The Flaveria bidentis beta-carbonic anhydrase gene family encodes cytosolic and chloroplastic isoforms demonstrating distinct organ-specific expression patterns.

Carbonic anhydrase (CA) catalyzes the interconversion of CO(2) and bicarbonate, the forms of inorganic carbon used by the primary carboxylating enzymes of C(3) and C(4) plants, respectively. Multiple forms of CA are found in both photosynthetic subtypes; however, the number of isoforms and the location and function of each have not been elucidated for any single plant species. Genomic Southern analyses showed that the C(4) dicotyledon Flaveria bidentis 'Kuntze' contains a small gene family encoding beta-CA and cDNAs encoding three distinct beta-CAs, named CA1, CA2, and CA3, were isolated. Quantitative reverse transcription-polymerase chain reactions showed that each member of this beta-CA family has a specific expression pattern in F. bidentis leaves, roots, and flowers. CA3 transcripts were at least 50 times more abundant than CA2 or CA1 transcripts in leaves. CA2 transcripts were detected in all organs examined and were the most abundant CA transcripts in roots. CA1 mRNA levels were similar to those of CA2 in leaves, but were considerably lower in roots and flowers. In vitro import assays showed CA1 was imported into isolated pea (Pisum sativum) chloroplasts, whereas CA2 and CA3 were not. These results support the following roles for F. bidentis CAs: CA3 is responsible for catalyzing the first step in the C(4) pathway in the mesophyll cell cytosol; CA2 provides bicarbonate for anapleurotic reactions involving nonphotosynthetic forms of phosphoenolpyruvate carboxylase in the cytosol of cells in both photosynthetic and nongreen tissues; and CA1 carries out nonphotosynthetic functions demonstrated by C(3) chloroplastic beta-CAs, including lipid biosynthesis and antioxidant activity.

Screening marine fungi for inhibitors of the C4 plant enzyme pyruvate phosphate dikinase: unguinol as a potential novel herbicide candidate.

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.

Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts.

Several radioreceptor assays using tritiated saxitoxin ([(3)H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.

Full length nucleotide sequence of a factor V-like subunit of oscutarin from Oxyuranus scutellatus scutellatus (coastal Taipan).

An Oxyuranus scutellatus scutellatus venom gland cDNA expression library was screened with antivenom. Positive clones were isolated and their nucleotide sequences determined. The complete sequence for a Factor V-like component from the Taipan venom prothrombin activator, oscutarin (EC 3.4.21.60) (Walker, F.J., Owen, W.G., Esmon, C.T., 1980. Characterization of the prothrombin activator from the venom of Oxyuranus scutellatus scutellatus (Taipan venom). Biochemistry, 19(5), 1020-1023; Speijer, H.G.R., Zwall, J., Robert, F.A., Rosing, J., 1986. Prothrombin acitvation by an activator from the venom of Oxyuranus Scutellatus (Taipan Snake). J. Biol. Chem. 261, 13258-13267) was determined from the sequencing of antigenic cDNA clones. The cDNA sequence encoded a protein of 1460 amino acid residues, including a 30-residue signal peptide. This sequence shared 95% sequence similarity with the non-enzymic subunit of the prothrombin activator (pseutarin C) from brown snake (Pseudonaja textilis) venom. This sequence in turn has been reported to share high similarity with mammalian Factor V. Sequence comparisons indicated the size, charge and cleavage sites were conserved across the two species. This is the first nucleotide sequence of a Factor V-like component from Oxyuranus venom and the second sequence within Elapidae to be reported.

Translation of in vitro inhibition by marine natural products of the C4 acid cycle enzyme pyruvate P(i) dikinase to in vivo C4 plant tissue death.

Marine organism derived extracts, previously identified as containing compounds that inhibited the C4 acid cycle enzyme pyruvate P(i) dikinase (PPDK), were assessed for their ability to exhibit an effect on the C4 plants Digitaria ciliaris and Echinochloa crus-galli. Oxygen electrode studies revealed that over half of these extracts inhibited C4 acid driven photosynthesis in leaf slices. Seventeen extracts had a deleterious effect on C4 plants in vivo within 24 h, whereas 36 caused an observable phytotoxic response in one or both of the C4 plants used for in vivo testing. None of the extracts inhibited PPDK metabolism of pyruvate via a directly competitive mechanism, instead hindering the enzyme by either mixed or uncompetitive means. This screening strategy, using a suite of assays, led to the isolation and identification of the herbicidal marine natural product ilimaquinone.

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides.

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.