Metabolism of naphthalene, 1-naphthol, indene, and indole by Rhodococcus sp strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently, Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphtalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation, However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound, Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer, Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

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.

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.

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P

Arsenic as a food chain contaminant:mechanisms of plant uptake and metabolism and mitigation strategies

Arsenic (As) is an environmental and food chain contaminant. Excessive accumulation of As, particularly inorganic arsenic (As(i)), in rice (Oryza sativa) poses a potential health risk to populations with high rice consumption. Rice is efficient at As accumulation owing to flooded paddy cultivation that leads to arsenite mobilization, and the inadvertent yet efficient uptake of arsenite through the silicon transport pathway. Iron, phosphorus, sulfur, and silicon interact strongly with As during its route from soil to plants. Plants take up arsenate through the phosphate transporters, and arsenite and undissociated methylated As species through the nodulin 26-like intrinsic (NIP) aquaporin channels. Arsenate is readily reduced to arsenite in planta, which is detoxified by complexation with thiol-rich peptides such as phytochelatins and/or vacuolar sequestration. A range of mitigation methods, from agronomic measures and plant breeding to genetic modification, may be employed to reduce As uptake by food crops.

Arsenic uptake and metabolism in plants

Arsenic (As) is an element that is nonessential for and toxic to plants. Arsenic contamination in the environment occurs in many regions, and, depending on environmental factors, its accumulation in food crops may pose a health risk to humans.Recent progress in understanding the mechanisms of As uptake and metabolism in plants is reviewed here. Arsenate is taken up by phosphate transporters. A number of the aquaporin nodulin26-like intrinsic proteins (NIPs) are able to transport arsenite,the predominant form of As in reducing environments. In rice (Oryza sativa), arsenite uptake shares the highly efficient silicon (Si) pathway of entry to root cells and efflux towards the xylem. In root cells arsenate is rapidly reduced to arsenite, which is effluxed to the external medium, complexed by thiol peptides or translocated to shoots. One type of arsenate reductase has been identified, but its in planta functions remain to be investigated. Some fern species in the Pteridaceae family are able to hyperaccumulate As in above-ground tissues. Hyperaccumulation appears to involve enhanced arsenate uptake, decreased arsenite-thiol complexation and arsenite efflux to the external medium, greatly enhanced xylem translocation of arsenite, and vacuolar sequestration of arsenite in fronds. Current knowledge gaps and future research directions are also identified.

Arsenic accumulation and metabolism in rice (Oryza sativa L.)

The use of arsenic (As) contaminated groundwater for irrigation of crops has resulted in elevated concentrations of arsenic in agricultural soils in Bangladesh, West Bengal (India), and elsewhere. Paddy rice (Oryza sativa L.) is the main agricultural crop grown in the arsenic-affected areas of Bangladesh. There is, therefore, concern regarding accumulation of arsenic in rice grown those soils. A greenhouse study was conducted to examine the effects of arsenic-contaminated irrigation water on the growth of rice and uptake and speciation of arsenic. Treatments of the greenhouse experiment consisted of two phosphate doses and seven different arsenate concentrations ranging from 0 to 8 mg of As L(-1) applied regularly throughout the 170-day post-transplantation growing period until plants were ready for harvesting. Increasing the concentration of arsenate in irrigation water significantly decreased plant height, grain yield, the number of filled grains, grain weight, and root biomass, while the arsenic concentrations in root, straw, and rice husk increased significantly. Concentrations of arsenic in rice grain did not exceed the food hygiene concentration limit (1.0 mg of As kg(-1) dry weight). The concentrations of arsenic in rice straw (up to 91.8 mg kg(-1) for the highest As treatment) were of the same order of magnitude as root arsenic concentrations (up to 107.5 mg kg(-1)), suggesting that arsenic can be readily translocated to the shoot. While not covered by food hygiene regulations, rice straw is used as cattle feed in many countries including Bangladesh. The high arsenic concentrations may have the potential for adverse health effects on the cattle and an increase of arsenic exposure in humans via the plant-animal-human pathway. Arsenic concentrations in rice plant parts except husk were not affected by application of phosphate. As the concentration of arsenic in the rice grain was low, arsenic speciation was performed only on rice straw to predict the risk associated with feeding contaminated straw to the cattle. Speciation of arsenic in tissues (using HPLC-ICP-MS) revealed that the predominant species present in straw was arsenate followed by arsenite and dimethylarsinic acid (DMAA). As DMAA is only present at low concentrations, it is unlikely this will greatly alter the toxicity of arsenic present in rice.

Changes in natural killer cells, the CD57CD8 subset, and related cytokines in healthy aging

Aging has been shown to be accompanied by various changes in the lymphocyte subset distribution in the elderly. We have investigated more fully, and in a large number of subjects, age-related changes within several subpopulations bearing natural killer (NK) cell-associated surface antigens and changes in several cytokines involved in NK cell expansion. A total of 229 healthy subjects from all decades of life from 20 to 98 years of age was included in this cross-sectional study. A significant increase with age was found in both the absolute counts and the proportions of CD3-CD(16+56)+, CD3+CD(16+56)+, CD57+CD8+, CD57+CD8(low)+, and CD57+CD8- cells, whereas the CD57+CD8(high)+ subset, which may represent the cytolytic T cell population more precisely, showed less change with age. Some evidence is also provided to suggest that these expanded NK cell populations are in an activated state. Soluble IL-2 receptor levels were also found to increase significantly with age and correlated with certain NK cell subsets. Although the functions of some of these subsets remain to be elucidated, their expansion in the elderly may represent a remodeling of the immune system with increasing age, with an increase in non-MHC-restricted cells perhaps compensating for the previously reported decline in T and B cells in the elderly. Alternatively, increased numbers of these cells may be a direct result of cytokine dysregulation or increased antigenic or neoplastic cell challenge.

Influences of phosphorus starvation on OsACR2.1 expression and arsenic metabolism in rice seedlings

The effects of phosphorus (P) status on arsenate reductase gene (OsACR2.1) expression, arsenate reductase activity, hydrogen peroxide (H(2)O(2)) content, and arsenic (As) species in rice seedlings which were exposed to arsenate after -P or +P pretreatments were investigated in a series of hydroponic experiments. OsACR2.1 expression increased significantly with decreasing internal P concentrations; more than 2-fold and 10-fold increases were found after P starvation for 30 h and 14 days, respectively. OsACR2.1 expression exhibited a significant positive correlation with internal root H(2)O(2) accumulation, which increased upon P starvation or exposure to H(2)O(2) without P starvation. Characterization of internal and effluxed As species showed the predominant form of As was arsenate in P-starved rice root, which contrasted with the +P pretreated plants. Additionally, more As was effluxed from P-starved rice roots than from non-starved roots. In summary, an interesting relationship was observed between P-starvation induced H(2)O(2) and OsACR2.1 gene expression. However, the up-regulation of OsACR2.1 did not increase arsenate reduction in P-starved rice seedlings when exposed to arsenate.

Serum Inflammatory Markers and Preeclampsia in Type 1 Diabetes:A prospective study

OBJECTIVE Inflammation and endothelial dysfunction have been associated with the immunobiology of preeclampsia (PE), a significant cause of adverse pregnancy outcomes. The prevalence of PE is elevated several fold in the presence of maternal type 1 diabetes mellitus (T1DM). Although cross-sectional studies of pregnancies among women without diabetes have shown altered inflammatory markers in the presence of PE, longitudinal studies of diabetic women are lacking. In maternal serum samples, we examined the temporal associations of markers of inflammation with the subsequent development of PE in women with T1DM. RESEARCH DESIGN AND METHODS We conducted longitudinal analyses of serum C-reactive protein (CRP), adhesion molecules, and cytokines during the first (mean ± SD, 12.2 ± 1.9 weeks), second (21.6 ± 1.5 weeks), and third (31.5 ± 1.7 weeks) trimesters of pregnancy (visits 1-3, respectively). All study visits took place before the onset of PE. Covariates were BMI, HbA1c, age of onset, duration of diabetes, and mean arterial pressure. RESULTS In women with T1DM who developed PE versus those who remained normotensive, CRP tended to be higher at visits 1 (P = 0.07) and 2 (P = 0.06) and was significantly higher at visit 3 (P <0.05); soluble E-selectin and interferon-?-inducible protein-10 (IP-10) were significantly higher at visit 3; interleukin-1 receptor antagonist (IL-1ra) and eotaxin were higher and lower, respectively, at visit 2 (all P <0.05). These conclusions persisted following adjustment for covariates. CONCLUSIONS In pregnant women with T1DM, elevated CRP, soluble E-selectin, IL-1ra, and IP-10 and lower eotaxin were associated with subsequent PE. The role of inflammatory factors as markers and potential mechanisms of the high prevalence of PE in T1DM merits further investigation.

Metabolism of very low- and low-density lipoproteins isolated from normolipidaemic type 2 (non-insulin-dependent) diabetic patients by human monocyte-derived macrophages

The very low- and low-density lipoprotein fractions were isolated from 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients in good to fair glycaemic control and from corresponding age-, sex-, and race-matched, non-diabetic control subjects. Rates of cholesteryl ester synthesis averaged 268 +/- 31 vs 289 +/- 40 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for very low- and 506 +/- 34 vs 556 +/- 51 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for low-density lipoproteins isolated from the Type 2 diabetic patients and control subjects, respectively, when they were incubated with human macrophages. A group of approximately one-third of the patients was selected for separate analyses because very low-density lipoproteins isolated from these patients did stimulate more cholesteryl ester synthesis when incubated with macrophages. There were no significant differences in the lipid composition of the lipoproteins isolated from the three groups of subjects. The relative proportion of apoprotein C to apoprotein E was significantly decreased (p less than 0.002) in the very low-density lipoproteins from diabetic patients and was further decreased in samples from these selected diabetic patients. The apoprotein C-I content of very low-density lipoproteins isolated from diabetic patients was increased compared to control subjects and was further increased in samples from the selected diabetic patients (p less than 0.02). There were no significant differences in the proportions of apoproteins C-III-0, C-III-1, or C-III-2 among the three groups. These studies suggest that in normolipidaemic Type 2 diabetic patients, the apoprotein composition of VLDL is abnormal and this may alter VLDL macrophage interactions and thus contribute to the increased prevalence of atherosclerosis in diabetic patients.