Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design. (C) 2002 Elsevier Science (USA). All rights reserved.

Zeolite/rock phosphate - a novel slow release phosphorus fertiliser for potted plant production

A glasshouse study was undertaken to determine if the zeolite mineral clinoptilolite from an Australian deposit in combination with rock phosphate (RP) could significantly enhance the uptake of P by sunflowers. The zeolite/RP combination was intended to act as an exchange-fertiliser, with Ca2+ exchanging onto the zeolite in response to plant uptake of nutrient cations (NH4+ or K) enhancing the dissolution of the RP. A reactive RP (Sechura) and a relatively non-reactive RP (Duchess) were examined. Zeolite was used in Ca2+-, K+- and NH4+-saturated forms at ratios of 3.5:1 and 7:1 with RP; Ca2+-zeolite was considered the control, with exchange-induced dissolution possible from K+-and NH4+-zeolite, The zeolite/RP mixture was applied as a vertical band adjacent to the sunflower seedling. In addition, N was supplied as urea in an effort to determine if RP dissolution resulted from H+ release by nitrification. Phosphorus supply from the zeolite/RP system was compared with an available P source (KH2PO4). The experiment clearly demonstrated greatly enhanced plant uptake of P from RP when applied in combination with NH4-zeolite, though the P uptake was lower than that from the soluble P source. The zeolite/RP interaction was much more effective with the reactive R-P than the non-reactive material, Within the NH4+-zeolite/RP band, root proliferation was greatly increased, as would be expected in an exchange-fertiliser system. The K+-zeolite system did not produce a significantly greater yield than the Ca2+-zeolite control, probably because adequate K+ supply from the basal application reduced uptake within the zeolite/RP band, thus reducing the extent of exchange-induced dissolution. Nevertheless, increased root proliferation within the band was observed, implying that exchange-induced dissolution may also be possible from this system. The zeolite/RP system offers the considerable advantage of P release in response to plant demand and is unique in this regard. (C) 2002 Elsevier Science B.V. All rights reserved.

Porphyrin profiles in blood and urine as a biomarker for exposure to various arsenic species

A sensitive method using HPLC with fluorescence detection has been established for the measurement of porphyrins in biological materials. The assay recoveries were 88.0 +/- 1.8% for protoporphyrin IX in the blood, and ranged from 98.3 +/- 2.7% to 111.1 +/- 7.4% for various porphyrins in the urine. This method was employed to investigate the altered porphyrin profiles in rats after a single dose of various arsenicals including soluble sodium arsenate and sodium arsenite, and the relatively insoluble calcium arsenite, calcium arsenate and arsenic-contaminated soils at dose rates of 5 mg/kg or 0.5 mg/kg body weight. Porphyrin concentrations increased within 24-48hr after the arsenic treatment in blood and urine. Protoporphyrin IX is the predominant porphyrin in the blood. In rats administered 5 mg As(III)/kg body weight, protoporphyrin IX concentration elevated to 123% of them control values in rats, 24 hr after the treatment. Higher increases were recorded in the urinary protoporphyrin IX (253% at 24 hr; 397% on day 2), uroporphyrin (121% at 24 hr; 208% on day 2) and coproporphyrin 111 (391% at 24 hr; 304% on day 2), while there was no significant increase (109% on day 3) observed in the urinary coproporphyrin I excretion. In rats administered 5 mg As(V)/kg, urinary excretion of protoporphyrin IX, uroporphyrin, coproporphyrin Ill and coproporphyrin I elevated to the maximum levels by 48 hr with the corresponding percentage values compared to the control being 177%, 158%, 224% and 143%, respectively. In rats dosed with 5 mg As(III)/kg, the increases (expressed as % of the control values) of protoporphyrin IX in the blood were in the order: sodium arsenite (144%) > sodium arsenate (125%) greater than or equal to calcium arsenite (123%) > calcium arsenate. In contrast, there was no significant increase of protoporphyrin K when the six arsenic-contaminated cattlei dip soils and nine copper chrome arsenate (CCA-contaminated) soils were administered to the rats. Probable explanations are discussed.

Characteristic zirconium-rich coring structures in Mg-Zr alloys

The most characteristic feature of the microstructure of a magnesium alloy that contains more than a few tenths per cent soluble zirconium is the zirconium-rich cores that exist in most grains. The morphology, distribution and composition of cores observed in a Mg-0.56%Zr alloy and the small particles present in them were investigated. (C) 2002 Acta Materialia Inc. Published by Elsevier Science Ltd. All rights reserved.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Hydrofluoric acid pre-treatment for improving 13C CPMAS NMR spectral quality of forest soils in south-east Queensland, Australia

Hydrofluoric acid (HF) was used to pre-treat forest soils of south-east Queensland for assessing the effectiveness of iron (Fe) removal, carbon (C) composition using C-13 cross-polarisation (CP) with magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) before and after the HF pre-treatment, and the improvement of C-13 CPMAS NMR spectra. Soil samples were collected from 4 experimental sites of different soil types, harvest residue management or prescribed burning, and tree species. More than 86% of Fe was in all soil types removed by the HF treatment. The C-13 NMR spectral quality was improved with increased resolution, especially in the alkyl C and O-alkyl C regions, and reduced NMR run-time (1-5 h per sample compared with >20 h per sample without the pre-treatment). The C composition appeared to alter slightly after the pre-treatment, but this might be largely due to improved spectrometer conditions and increased resolution leading to more accurate NMR spectral integration. Organic C recovery after HF pre-treatment varied with soil types and forest management, and soluble soil organic matter (SOM) could be lost during the pre-treatment. The Fourier Transform-Infrared (FT-IR) spectra of HF extracts indicated the preferential removal of carboxylic C groups during the pre-treatment, but this could also be due to adsorbed water on the mineral matter. The NMR spectra revealed some changes in C composition and quality due to residue management and decomposition. Overall, the HF treatment was a useful pre-treatment for obtaining semi-quantitative C-13 CPMAS NMR spectra of subtropical Australian forest soils.

Identification of micronutrient deficiency of wheat in the Peshawar Valley, Pakistan

A field experiment was conducted to study the effect of micronutrients, zinc (Zn), copper (Cu), iron (Fe), manganese (Mn), boron (13) and a commercial fritted micronutrient product called Zarzameen, on the yield and the yield components of wheat (Triticum aestivum L.), in the Peshawar valley, Pakistan. Different combinations of Zn, Cu. Fe. Mn, B, and Zarzameen were applied at the rate of 4.0, 2.0, 5.0, 2.0, 1.0 kg ha(-1) and 1.0 kg ha(-1), respectively, along with a basal dose of 100 kg ha(-1) nitrogen(N), 75 kg ha(-1) phosphorus (P) and 50 kg ha(-1) potassium (K). The fertilizer treatments (macro- and micronutrients) increased wheat dry matter, grain yield, and straw yield significantly over an unfertilized control. Soil tests for B and Zn were increased both at boot and harvesting stage, and Fe at boot stage, with the addition of micronutrients. Plants without B had showed classical B deficiency symptoms at grain formation stage, but not at vegetative stage. Boron concentration in the dry matter of wheat plants increased with the addition of the B fertilizer in the soil. Boron deficiency was not observed in plants containing >4 mg B kg(-1) at the boot stage, or in soils containing > 1.4 mg kg(-1) hot water soluble B.

Starch degradation in 'Kensington' mango fruit following heat treatments

Changes in carbohydrate metabolism of 'Kensington' mango fruit from 2 major production regions in Queensland were measured after conditioning fruit with hot air at 40degreesC for 0, 2, 4, 8 and 16 h or at 22degreesC for 16 h (control) followed by hot-water treatment at either 45degreesC fruit-core temperature for 30 min or 47degreesC fruit-core temperature held for 15 min. Advancing physiological maturity of 'Kensington' mango fruit was correlated with increased starch concentration within the mesocarp. An alpha-amylase inhibitor was present in unripe 'Kensington' mesocarp. alpha-Amylase activity was promoted by conditioning fruit at 40degreesC for 8 h, and this enhanced enzyme activity persisted until the fruit were ripe. Consequently, starch degradation was accelerated and the concentration of total soluble solids was higher in fruit conditioned at 40degreesC for 8 h than in fruit left at the lower temperature of 22degreesC for 16 h or not conditioned. Immediately on removal of fruit from hot-water treatment, activities of alpha-amylase and phosphorylase were inhibited. This inhibition was correlated with higher starch concentration and starch layer and starch spot injuries in these fruit. A positive correlation was also found between increased sucrose concentration and greater starch loss in 40degreesC conditioned 'Kensington' fruit. It is proposed that increased sugar concentration in the mesocarp increased the level of fruit heat tolerance.

Tracing the fate of N-15-enriched feed in an intensive shrimp system

The fate of N-15-nitrogen-enriched formulated feed fed to shrimp was traced through the food web in shallow, outdoor tank systems (1000 1) stocked with shrimp. Triplicate tanks containing shrimp water with and without sediment were used to identify the role of the natural biota in the water column and sediment in processing dietary nitrogen (N). A preliminary experiment demonstrated that N-15-nitrogen-enriched feed products could be detected in the food web. Based on this, a 15-day experiment was conducted. The ammonium (NH4+) pool in the water column became rapidly enriched (within one day) with N-15-nitrogen after shrimp were fed N-15-enriched feed. By day 15, 6% of the added N-15-nitrogen was in this fraction in the 'sediment' tanks compared with 0.4% in the 'no sediment' tanks. The particulate fraction in the water column, principally autotrophic nanoflagellates, accounted for 4-5% of the N-15-nitrogen fed to shrimp after one day. This increased to 16% in the 'no sediment' treatment, and decreased to 2% in the 'sediment' treatment by day 15. It appears that dietary N was more accessible to the phytoplankton community in the absence of sediment. The difference is possibly because a proportion of the dietary N was buried in the sediment in the 'sediment' treatment, making it unavailable to the phytoplankton. Alternatively, the dietary N was retained in the NH4+ pool in the water column since phytoplankton growth, and hence, N utilization was lower in the 'sediment' treatment. The lower growth of phytoplankton in the 'sediment' treatment appeared to be related to higher turbidity, and hence, lower light availability for growth. The percentage N-15-nitrogen detected in the sediment was only 6% despite the high capacity for sedimentation of the large biomass of plankton detritus and shrimp waste. This suggests rapid remineralization of organic waste by the microbial community in the sediment resulting in diffusion of inorganic N sources into the water column. It is likely that most of the dietary N will ultimately be removed from the tank system by water discharges. Our study showed that N-15-nitrogen derived from aquaculture feed can be processed by the microbial community in outdoor aquaculture systems and provides a method for determining the effect of dietary N on ecosystems. However, a significant amount of the dietary N was not retained by the natural biota and is likely to be present in the soluble organic fraction. (C) 2002 Elsevier Science B.V. All rights reserved.

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Effect of soil composition and dissolved organic matter on pesticide sorption

The effect of the solid and dissolved organic matter fractions, mineral composition and ionic strength of the soil solution on the sorption behaviour of pesticides were studied. A number of soils, chosen so as to have different clay mineral and organic carbon content, were used to study the sorption of the pesticides atrazine (6-chloro-N-2-ethyl-N-4-isopropyl-1,3,5-triazine-2,4-diamine), 2,4-D ((2,4-dichlorophenoxy) acetic acid), isoproturon (3-(4-isopropylphenyl)1,1-dimethylurea) and paraquat (1,1'-dimethyl-4,4'-bipyridinium) in the presence of low and high levels of dissolved organic carbon and different background electrolytes. The sorption behaviour of atrazine, isoproturon and paraquat was dominated by the solid state soil components and the presence of dissolved organic matter had little effect. The sorption of 2,4-D was slightly affected by the soluble organic matter in the soil. However, this effect may be due to competition for adsorption sites between the pesticide and the soluble organic matter rather than due to a positive interaction between the pesticide and the soluble fraction of soil organic matter. It is concluded that the major factor governing the sorption of these pesticides is the solid state organic fraction with the clay mineral content also making a significant contribution. The dissolved organic carbon fraction of the total organic carbon in the soil and the ionic strength of the soil solution appear to have little or no effect on the sorption/transport characteristics of these pesticides over the range of concentrations studied. (C) 2002 Elsevier Science B.V. All rights reserved.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Identification of intracellular calcium oxalate crystals in Chamelaucium uncinatum (Myrtaceae)

Intracellular inclusions in the pedicel and calyx-tube tissues of Chamelaucium uncinatum Schauer ( Myrtaceae) flowers are irregular in shape. They were shown, by polarised light and scanning electron microscopy, to be birefringent 8.9-29.5 mum druse (i.e. aggregate) crystals. Energy-dispersive X-ray spectroscopy showed that these crystals were predominantly composed of calcium. Histochemical and acid-solubility tests indicated that the crystals were calcium oxalate. Raman microprobe spectroscopy was used to confirm this chemical identity. The calcium oxalate crystals were located in xylem-vessel lumens and also in parenchyma cells adjacent to vascular tissues. Thus, the crystals may function to regulate soluble calcium concentrations in C. uncinatum tissues near sites where calcium is unloaded from the xylem.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.

The mouse secretome: functional classification of the proteins secreted into the extracellular environment

We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins