Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [H-2(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats pi and 0.1 mg pyridoxine (PN)/kg diet]. [H-2(3)]Leucine and [1-C-13]methionine were also used to examine turnover of protein and methionine pools, respectively, All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [H-2(3)]leucine. Hepatic [H-2(2)]methionine production from [H-2(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [H-2(3)]cysteine from the [H-2(3)]serine increased over twofold. In contrast, plasma appearance of [H-2(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-C-13]homocysteine/[1-C-13]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

Utilization of organophosphonates by environmental microorganisms

A survey of the utilization by environmental micro-organisms of a range of compounds containing the carbon-phosphorus (C-P) bond was carried out. Elective culture studies indicated that 15 of 19 alkylphosphonates tested served only as a sole source of phosphorus for microbial growth. Their metabolism did not lead to the extracellular release of inorganic phosphate. However, four organophosphonates - phosphonoacetate, phosphonoalanine, 2-aminoethylphosphonate and phosphonomycin - supported microbial growth when supplied as either a phosphorus source or as a carbon and energy source, with near-quantitative inorganic phosphate release. Four of five amino alkylphosphonates tested were also utilized as a nitrogen source in the presence of 1 mmol l(-1) inorganic phosphate. In a subsequent screening programme, 99% of bacterial isolates tested were able to utilize 2-aminoethylphosphonate as a sole phosphorus source, 61% as a nitrogen source, 10% as a source of nitrogen and phosphorus, and 2% as a source of carbon, nitrogen and phosphorus; 2% of isolates used phosphonoalanine as a nitrogen source. These results suggest that the uptake and metabolism of organophosphonates by bacteria is less 'tightly' regulated by phosphorus starvation than has previously been supposed.

Biodegradation of phosphonomycin by Rhizobium huakuii PMY1

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phasphonomycin as a carbon, energy, and phosphorus source with accompanying P-1 release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon-phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase: represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it: is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l(-1) and 100 mmol l(-1), respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity ibr this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.

Deregulated estrogen metabolism in BRCA1 deficient cells induces chromosomal instability.

Objectives: Germline mutations in BRCA1 predispose carriers to a high
incidence of breast and ovarian cancers. The BRCA1 protein functions to maintain
genomic stability via important roles in DNA repair, transcriptional regulation, and
post-replicative repair. Despite functions in processes essential in all cells, BRCA1
loss or mutation leads to tumours predominantly in estrogen-regulated tissues.
Here, we aim to determine if endogenous estrogen metabolites may be an initiator
of genomic instability in BRCA1 deficient cells.

Methods: We analysed DNA DSBs by ?H2AX, 53BP1, and pATM1981
foci and neutral comet assay, estrogen metabolite concentrations by LC-MS/MS,
and BRCA1 transcriptional regulation of metabolism genes by ChIP-chip, ChIP,
and qRT-PCR.

Results: We show that estrogen metabolism is perturbed in BRCA1 deficient
cells resulting in elevated production of 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), and decreased production of the protective metabolite
4-methoxyestradiol. We demonstrate that 2-OHE2 and 4-OHE2 treatment leads
to DNA double strand breaks (DSBs) in breast cells, and these DSBs were exacerbated
in both BRCA1 depleted cells and BRCA1 heterozygous cells (harbouring
185delAG mutation). Furthermore, the DSBs were not repaired efficiently in either
BRCA1 depleted or heterozygous cells, and we found that 2-OHE2 and 4-OHE2
treatment generates chromosomal aberrations in BRCA1 depleted cells. We suggest
that the increase in DNA DSBs in BRCA1 deficient cells is due to loss of
both BRCA1 transcriptional repression of estrogen metabolising genes (such as
CYP1A1 and CYP3A4) and loss of transcriptional activation of detoxification
genes (such as COMT).

Conclusions: We suggest that BRCA1 loss results in estrogen driven tumourigenesis
through a combination of increased expression of estrogen metabolising
enzymes and reduced expression of protective enzymes, coupled with a defect in
the repair of DNA DSBs induced by endogenous estrogen metabolites. The overall
effect being an exacerbation of genomic instability in estrogen regulated tissues in
BRCA1 mutation carriers.

Reproductive biomass in Holcus lanatus clones that differ in their phosphate uptake kinetics and mycorrhizal colonization

In normal populations of the common grass Holcus lanatus there is a polymorphism for arsenate resistance, manifested as suppressed phosphate uptake (SPU), and controlled by a major gene with dominant expression. A natural population of SPU plants had greater arbuscular-mycorrhizal colonization than wild type, nonSPU plants. It was hypothesized that, in order to survive alongside plants with a normal rate of phosphate (P) uptake, SPU plants would be more dependent on mycorrhizal associations. We performed an experiment using plants with SPU phenotypes from both arsenate mine spoils and uncontaminated soils, as well as plants with a nonSPU phenotype. They were grown with and without a mycorrhizal inoculum and added N, which altered plant P requirements. We showed that grasses with SPU phenotypes accumulated more shoot P than nonSPU plants, the opposite of the expected result. SPY plants also produced considerably more flower panicles, and had greater shoot and root biomass. The persistence of SPU phenotypes in normal populations is not necessarily related to mycorrhizal colonization as there were no differences in percentage AM colonization between the phenotypes. Being mycorrhizal reduced flower biomass production, as mycorrhizal SPU plants had lower shoot P concentrations and produced fewer flower panicles than non-mycorrhizal, nonSPU plants. We now hypothesize that the SPU phenotype is brought about by a genotype that results in increased accumulation of P in shoots, and that suppression of the rate of uptake is a consequence of this high shoot P concentration, operating by means of a homeostatic feedback mechanism. We also postulate that increased flower production is linked to a high shoot P concentration. SPU plants thus allocate more resources into seed production, leading to a higher frequency of SPU genes. Increased reproductive allocation reduces vegetative allocation and may affect competitive ability and hence survival, explaining the maintenance of the polymorphism. As mycorrhizal SPU plants behave more like nonSPU plants, AM colonization itself could play a major part in the maintenance of the SPU polymorphism.

An arsenic-accumulating, hypertolerant brassica, Isatis capadocica

Isatis capadocica, a brassica collected from Iranian arsenic-contaminated mine spoils and control populations, was examined to determine arsenate tolerance, metabolism and accumulation. I. cappadocica exhibited arsenate hypertolerance in both mine and nonmine populations, actively growing at concentrations of > 1 mm arsenate in hydroponic solution. I. cappadocica had an ability to accumulate high concentrations of arsenic in its shoots, in excess of 100 mg kg(-1) DW, with a shoot : root transfer ratio of > 1. The ability to accumulate arsenic was exhibited in both hydroponics and contaminated soils. Tolerance in this species was not achieved through suppression of high-affinity phosphate/arsenate root transport, in contrast to other monocotyledons and dicotyledons. A high percentage (> 50%) of arsenic in the tissues was phytochelatin complexed; however, it is argued that this is a constitutive, rather than an adaptive, mechanism of tolerance.

Rice-arsenate interactions in hydroponics:whole genome transcriptional analysis

Rice (Oryza sativa) varieties that are arsenate-tolerant (Bala) and -sensitive (Azucena) were used to conduct a transcriptome analysis of the response of rice seedlings to sodium arsenate (AsV) in hydroponic solution. RNA extracted from the roots of three replicate experiments of plants grown for 1 week in phosphate-free nutrient with or without 13.3 muM AsV was used to challenge the Affymetrix (52K) GeneChip Rice Genome array. A total of 576 probe sets were significantly up-regulated at least 2-fold in both varieties, whereas 622 were down-regulated. Ontological classification is presented. As expected, a large number of transcription factors, stress proteins, and transporters demonstrated differential expression. Striking is the lack of response of classic oxidative stress-responsive genes or phytochelatin synthases/synthatases. However, the large number of responses from genes involved in glutathione synthesis, metabolism, and transport suggests that glutathione conjugation and arsenate methylation may be important biochemical responses to arsenate challenge. In this report, no attempt is made to dissect differences in the response of the tolerant and sensitive variety, but analysis in a companion article will link gene expression to the known tolerance loci available in the BalaxAzucena mapping population.

Rice-arsenate interactions in hydroponics: a three-gene model for tolerance

In this study, the genetic mapping of the tolerance of root growth to 13.3 muM arsenate [As(V)] using the BalaxAzucena population is improved, and candidate genes for further study are identified. A remarkable three-gene model of tolerance is advanced, which appears to involve epistatic interaction between three major genes, two on chromosome 6 and one on chromosome 10. Any combination of two of these genes inherited from the tolerant parent leads to the plant having tolerance. Lists of potential positional candidate genes are presented. These are then refined using whole genome transcriptomics data and bioinformatics. Physiological evidence is also provided that genes related to phosphate transport are unlikely to be behind the genetic loci conferring tolerance. These results offer testable hypotheses for genes related to As(V) tolerance that might offer strategies for mitigating arsenic (As) accumulation in consumed rice.

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics, interactions with phosphate, and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).

Development of a bovine collagen-apatitic calcium phosphate cement for potential fracture treatment through vertebroplasty

The aim of this study was to examine the potential of incorporating bovine fibres as a means of reinforcing a typically brittle apatite calcium phosphate cement for vertebroplasty. Type I collagen derived from bovine Achilles tendon was ground cryogenically to produce an average fibre length of 0.96 ± 0.55 mm and manually mixed into the powder phase of an apatite-based cement at 1, 3 or 5 wt.%. Fibre addition of up to 5 wt.% had a significant effect (P = 0.001) on the fracture toughness, which was increased by 172%. Adding =1 wt.% bovine collagen fibres did not compromise the compressive properties significantly, however, a decrease of 39-53% was demonstrated at =3 wt.% fibre loading. Adding bovine collagen to the calcium phosphate cement reduced the initial and final setting times to satisfy the clinical requirements stated for vertebroplasty. The cement viscosity increased in a linear manner (R = 0.975) with increased loading of collagen fibres, such that the injectability was found to be reduced by 83% at 5 wt.% collagen loading. This study suggests for the first time the potential application of a collagen-reinforced calcium phosphate cement as a viable option in the treatment of vertebral fractures, however, issues surrounding efficacious cement delivery need to be addressed. © 2012 Acta Materialia Inc.

Development of calcium phosphate cement for the augmentation of traumatically fractured porcine specimens using vertebroplasty

The study aim was to develop and apply an experimental technique to determine the biomechanical effect of polymethylmethacrylate (PMMA) and calcium phosphate (CaP) cement on the stiffness and strength of augmented vertebrae following traumatic fracture. Twelve burst type fractures were generated in porcine three-vertebra segments. The specimens were randomly split into two groups (n=6), imaged using microCT and tested under axial loading. The two groups of fractured specimens underwent a vertebroplasty procedure, one group was augmented with CaP cement designed and developed at Queen's University Belfast. The other group was augmented with PMMA cement (WHW Plastics, Hull, UK). The specimens were imaged and re-tested . An intact single vertebra specimen group (n=12) was also imaged and tested under axial loading. A significant decrease (p<0.01) was found between the stiffness of the fractured and intact groups, demonstrating that the fractures generated were sufficiently severe, to adversely affect mechanical behaviour. Significant increase (p<0.01) in failure load was found for the specimen group augmented with the PMMA cement compared to the pre-augmentation group, conversely, no significant increase (p<0.01) was found in the failure load of the specimens augmented with CaP cement, this is attributed to the significantly (p<0.05) lower volume of CaP cement that was successfully injected into the fracture, compared to the PMMA cement. The effect of the percentage of cement fracture fill, cement modulus on the specimen stiffness and ultimate failure load could be investigated further by using the methods developed within this study to test a more injectable CaP cement.