Flow-directed PCA for monitoring networks.

Measurements recorded over monitoring networks often possess spatial and temporal correlation inducing redundancies in the information provided. For river water quality monitoring in particular, flow-connected sites may likely provide similar information. This paper proposes a novel approach to principal components analysis to investigate reducing dimensionality for spatiotemporal flow-connected network data in order to identify common spatiotemporal patterns. The method is illustrated using monthly observations of total oxidized nitrogen for the Trent catchment area in England. Common patterns are revealed that are hidden when the river network structure and temporal correlation are not accounted for. Such patterns provide valuable information for the design of future sampling strategies.

Nucleotides of tRNA (Glu) involved in recognition by barley chloroplast glutamyl-tRNA synthetase and glutamyl-tRNA reductase.

The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex with the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the recognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a number of species were used to determine the nucleotides involved in the recognition of the barley chloroplast tRNA(Glu) by the barley enzymes. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The chemically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA synthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chloroplasts if barley, Chlamydomonas reinhardtii, tobacco, cucumber, wheat and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia coli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 conserved bases and five of these bases, G10, A26, U34, U35 and A37 are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(Glu) from E. coli, bakers yeast and barley germ and the barley chloroplast glutamyl-tRNA(Gln) are not effective substrates for the barley chloroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences of these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yields seven common differences in the primary sequence. These 7 nucleotides, A7-U66, U29-A41, A53-U61, and U72 are expected to be required for recognition by the barley chloroplast glutamyl-tRNA(Glu) reductase.

Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase.

In chlorophyll biosynthesis, insertion of Mg(2+) into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (R(free)=24.5 %). It belongs to the chaperone-like "ATPase associated with a variety of cellular activities" (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.

Spatiotemporal statistical modelling of long-term change in river nutrient concentrations in England & Wales.

Concentrations of nutrient nitrogen (N) and phosphorus (P) are elevated in rivers across large areas of Europe (European Nitrogen Assessment (ENA), Sutton et al., 2011). Environmental policies have been implemented over the past 20 years with the aim of reducing nitrogen inputs to surface waters. However, environmental and ecological status is still below set targets (ENA, Sutton et al., 2011). Identification of patterns in long-term change for nutrient trends in hydrological catchments in England & Wales is required to assess impacts of nutrient management policy and provide better evidence for future policy. Such information could provide essential evidence for supporting policy by combining information from the wider catchment, rather than relying on the analysis of data from individual sites. Surface water quality is subject to considerable spatial and short-period temporal variability, reflecting variability in loading and dilution. This makes it difficult to determine temporal trends at individual monitoring sites with relatively sparse sampling. Here we apply spatiotemporal statistical additive models for both nitrogen and phosphorus in river networks across England & Wales to investigate the overall pattern of nutrient concentrations in these river surface waters over the past 20-40 years. Concentrations of Orthophosphate (OP) have generally decreased over time for many of the Large Hydrological Areas with a seasonal pattern highlighting one peak in the summer months. Over the past ten years, Total Oxidised Nitrogen (Nitrate+Nitrite, TON) concentrations have generally been slowly decreasing or fairly constant. However, prior to 2000, concentrations were generally on an upward trend. The seasonal pattern highlights one trough in the summer months. The highest levels for OP and TON broadly occur in the same general areas across England & Wales. On average, over time, the lowest values are evident in the north-west and south-west (particularly for OP) and highest values are evident in the Midlands, Anglian and Southern regions.

Distributed and dynamic modelling of hydrology, phosphorus and ecology in the Hampshire Avon and Blashford Lakes: evaluating alternative strategies to meet WFD standards.

The issues of diffuse and point source phosphorus (P) pollution in the Hampshire Avon and Blashford Lakes are explored using a catchment model of the river system. A multibranch, process based, dynamic water quality model (INCA-P) has been applied to the whole river system to simulate water fluxes, total phosphorus (TP) and soluble reactive phosphorus (SRP) concentrations and ecology. The model has been used to assess impacts of both agricultural runoff and point sources from waste water treatment plants (WWTPs) on water quality. The results show that agriculture contributes approximately 40% of the phosphorus load and point sources the other 60% of the load in this catchment. A set of scenarios have been investigated to assess the impacts of alternative phosphorus reduction strategies and it is shown that a combined strategy of agricultural phosphorus reduction through either fertiliser reductions or better phosphorus management together with improved treatment at WWTPs would reduce the SRP concentrations in the river to acceptable levels to meet the EU Water Framework Directive (WFD) requirements. A seasonal strategy for WWTP phosphorus reductions would achieve significant benefits at reduced cost.

Amenorrhea.

The appearance of amenorrhea relates to a wide variety of changes within the body. Some of these changes are simple in their cause and in their management. Others are complex and obscure. A classification of amenorrhea is discussed. An approach is described whereby the patient may be investigated and the degree of complexity determined. Some proposed cases are described to illustrate these principles.

Urinary estriol estimations in normal and abnormal pregnancies.

Urinary estriol estimations from 24-hour urine specimens were studied in 65 women in the course of normal pregnancies and compared to 18 analyses in women in whom fetal death had occurred. There is a significant difference in the levels of estriol excretion between these two categories. Serial studies were carried out on patients whose pregnancies ended in normal delivery and in patients whose pregnancies were complicated by eclampsia and by hypertension of various degrees of severity. A definite correlation between urinary estriol excretion and the health of the fetus in utero was found. The information that these estimations provide can be of assistance to the clinician in the management of selected patients where the fetus is in danger prior to delivery.

Mechanism and regulation of Mg-chelatase.

Mg-chelatase catalyses the insertion of Mg into protoporphyrin IX (Proto). This seemingly simple reaction also is potentially one of the most interesting and crucial steps in the (bacterio)chlorophyll (Bchl/Chl)-synthesis pathway, owing to its position at the branch-point between haem and Bchl/Chl synthesis. Up until the level of Proto, haem and Bchl/Chl synthesis share a common pathway. However, at the point of metal-ion insertion there are two choices: Mg2+ insertion to make Bchl/Chl (catalysed by Mg-chelatase) or Fe2+ insertion to make haem (catalysed by ferrochelatase). Thus the relative activities of Mg-chelatase and ferrochelatase must be regulated with respect to the organism's requirements for these end products. How is this regulation achieved? For Mg-chelatase, the recent design of an in vitro assay combined with the identification of Bchl-biosynthetic enzyme genes has now made it possible to address this question. In all photosynthetic organisms studied to date, Mg-chelatase is a three-component enzyme, and in several species these proteins have been cloned and expressed in an active form. The reaction takes place in two steps, with an ATP-dependent activation followed by an ATP-dependent chelation step. The activation step may be the key to regulation, although variations in subunit levels during diurnal growth may also play a role in determining the flux through the Bchl/Chl and haem branches of the pathway.

Heterologous expression of the Rhodobacter capsulatus BchI, -D, and -H genes that encode magnesium chelatase subunits and characterization of the reconstituted enzyme.

Magnesium chelatase inserts Mg2+ into protoporphyrin IX in the chlorophyll and bacteriochlorophyll biosynthetic pathways. In photosynthetic bacteria, the products of three genes, bchI, bchD, and bchH, are required for magnesium chelatase activity. These genes from Rhodobacter capsulatus were cloned separately into expression plasmids pET3a and pET15b. The pET15b constructs produced NH2-terminally His6-tagged proteins. All proteins were highly expressed and were purified to near homogeneity. The BchI and BchH proteins were soluble. BchD proteins were insoluble, inactive inclusion bodies that were renatured by rapid dilution from 6 M urea. The presence of BchI in the solution into which the urea solution of BchD was diluted increased the yield of active BchD. A molar ratio of 1 BchI:1 BchD was sufficient for maximum renaturation of BchD. All of the proteins were active in the magnesium chelatase assay except His-tagged BchI, which was inactive and inhibited in incubations containing non-His-tagged BchI. Expressed BchH proteins contained tightly bound protoporphyrin IX, and they were susceptible to inactivation by light. Maximum magnesium chelatase activity per mol of BchD occurred at a stoichiometry of 4 BchI:1 BchD. The optimum reaction pH was 8.0. The reaction exhibited Michaelis-Menten kinetics with respect to protoporphyrin IX and BchH.

Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803.

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100, 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.

EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer.

BchI, belonging to the AAA+ -protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA+ -protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA+ -proteins are Mg2+ -dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI-ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at approximately 30A and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30A.

Enzymic and mechanistic studies on the conversion of glutamate to 5-aminolaevulinate.

Higher plants, algae, cyanobacteria and several other photosynthetic and non-photosynthetic bacteria synthesize 5-aminolaevulinate by a tRNA(Glu)-mediated pathway. Glutamate is activated at the alpha-carboxyl by ligation to tRNA(Glu) with an aminoacyl-tRNA synthetase. An NADPH-dependent reductase converts glutamyl-tRNA(Glu) to glutamate 1-semialdehyde, which is finally converted to 5-aminolaevulinate by an aminotransferase. These components are soluble and in plants and algae are located in the chloroplast stroma. In plants and algae the tRNA(Glu) is encoded in chloroplast DNA whereas the enzymes are encoded in nuclear DNA. The tRNA(Glu) has a hypermodified 5-methylaminomethyl-2-thiouridine-pseudouridine-C anticodon and probably plays a role in the light-dark regulation of 5-aminolaevulinate synthesis. Ligation of glutamate to tRNA(Glu) requires ATP and Mg2+ and proceeds via a ternary intermediate. Glutamyl-tRNA(Glu) reduction appears to involve formation of a complex. Glutamate 1-semialdehyde non-enzymically synthesized by reductive ozonolysis from gamma-vinyl GABA is used as substrate by the last enzyme. Glutamate-1-semialdehyde aminotransferase contains pyridoxal phosphate as a prosthetic group. The enzyme is converted to spectrally different forms by treatment with 4,5-diaminovalerate or 4,5-dioxovalerate. The pyridoxamine 5'-phosphate form of the enzyme converts (S)-glutamate 1-semialdehyde to 5-aminolaevulinate via 4,5-diaminovalerate through a bi-bi ping-pong mechanism.

Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers.

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg(2+) into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA(+)) family of proteins. AAA(+) proteins are Mg(2+)-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.

Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit.

The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A.

Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides.

The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.

Phytobilin biosynthesis: the Synechocystis sp. PCC 6803 heme oxygenase-encoding ho1 gene complements a phytochrome-deficient Arabidopsis thalianna hy1 mutant.

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXalpha, in a reaction catalyzed by heme oxygenase. An Arabidopsis thaliana hy1 mutant was previously shown to be deficient in phytochrome responses, and these responses were regained when the plants were administered biliverdin IXalpha. A heme oxygenase-encoding gene, ho1, was recently cloned from the cyanobacterium Synechocystis sp. PCC 6803. When ho1 was expressed in Escherichia coli, the cells produced active ferredoxin-dependent soluble heme oxygenase. The open reading frame of ho1 was fused in frame with a chloroplast transit peptide-encoding sequence from the oli gene of Antirrhinum majus. This construct was placed in a binary plasmid vectorcontaining a kanamycin resistance marker and a cauliflower mosaic virus 35S promoter to control expression of the chimeric oli-ho1 gene and used to transform A. thaliana hy1 plants. Two independent transformed lines were obtained that had the phenotype of the parental Landsberg erecta line and expressed the chimeric gene, as indicated by detection of its mRNA by reverse transcriptase-polymerase chain reaction. The results indicate that Synechocystis sp. PCC 6803 heme oxygenase encoded by ho1 can substitute for the defective HY1 gene product and that the only required enzyme activity of the HY1 gene product is heme oxygenase.

Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.

Structural genes for Mg-chelatase subunits in barley: Xantha-f, -g and -h.

Barley mutants in the loci Xantha-f, Xantha-g and Xantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, the xan-f, -g and -h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein of Arabidopsis thaliana and the OLIVE (OLI) protein of Antirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. The xan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and the A. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from the xantha mutants and confirmed the results of the Western analysis. The mutants xan-f27, -f40, -h56 and -h57 are defective in transcript accumulation while -h38 is defective in translation. Southern blot analysis established that h38 has a deletion of part of the gene. Mutants xan-f10 and -f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that X an-f -h genes encode two subunits of the barley Mg-chelatase and that X an-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein of Antirrhinum, 66% to the Synechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS protein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA proteins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-chelatase. Identification of the barley X an-f and X an-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that the Antirrhinum OLI protein and the Arabidopsis Ch42 protein are subunits of Mg-chelatase in these plants. The expression of both thet X an-f and -h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.