Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues Conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to (he dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds Conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1.3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo ill a number of human tumor xenograft models.

Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations

Elevation in plasma homocysteine concentration has been associated with vascular disease and neural tube defects. Methionine synthase is a vitamin B(12)-dependent enzyme that catalyses the remethylation of homocysteine to methionine. Therefore, defects in this enzyme may result in elevated homocysteine levels. One relatively common polymorphism in the methionine synthase gene (D919G) is an A to G transition at bp 2,756, which converts an aspartic acid residue believed to be part of a helix involved in co-factor binding to a glycine. We have investigated the effect of this polymorphism on plasma homocysteine levels in a working male population (n = 607) in which we previously described the relationship of the C677T "thermolabile" methylenetetrahydrofolate reductase (MTHFR) polymorphism with homocysteine levels. We found that the methionine synthase D919G polymorphism is significantly (P = 0.03) associated with homocysteine concentration, and the DD genotype contributes to a moderate increase in homocysteine levels across the homocysteine distribution (OR = 1.58, DD genotype in the upper half of the homocysteine distribution, P = 0.006). Unlike thermolabile MTHFR, the homocysteine-elevating effects of the methionine synthase polymorphism are independent of folate and B(12) levels; however, the DD genotype has a larger homocysteine-elevating effect in individuals with low B(6) levels. This polymorphism may, therefore, make a moderate, but significant, contribution to clinical conditions that are associated with elevated homocysteine.

Effect of B-group vitamins and antioxidant vitamins on hyperhomocysteinemia:a double-blind, randomized, factorial-design, controlled trial

Mild hyperhomocysteinemia is accepted as a risk factor for premature cardiovascular disease. In a population with a high prevalence of cardiovascular disease, we screened a group of clinically healthy working men aged 30-49 y (n = 509) for plasma homocysteine and 5,10-methylene tetrahydrofolate reductase (MTHFR) genotype status. Those with mildly elevated homocysteine concentrations (> or = 8.34 micromol/L) were selected for intervention. In a randomized, factorial-design, controlled trial we assessed the effects of B-group vitamins and antioxidant vitamin supplementation on homocysteine concentrations. The 132 men were randomly assigned to one of four groups: supplementation with B-group vitamins alone (1 mg folic acid, 7.2 mg pyridoxine, and 0.02 mg cyanocobalamin), antioxidant vitamins alone (150 mg ascorbic acid, 67 mg RRR-alpha-tocopherol, and 9 mg beta-carotene), B-group vitamins with antioxidant vitamins, or placebo. Intervention was double-blind. A total of 101 men completed the 8-wk intervention. When homocysteine concentrations were analyzed by group, significant (P <0.001) decreases (32.0% and 30.0%, respectively) were observed in both groups receiving B-group vitamins either with or without antioxidants. The effect of B-group vitamins alone over 8 wk was a reduction in homocysteine concentrations of 27.9% (95% CI: 22.0%, 33.3%; P <0.001) whereas antioxidants alone produced a nonsignificant increase of 5.1% (95% CI: -2.8%, 13.6%; P = 0.21). There was no evidence of any interaction between the two groups of vitamins. The effect of B-group vitamin supplementation seemed to depend on MTHFR genotype. Supplementation with the B-group vitamins with or without antioxidants reduced homocysteine in the men with mildly elevated concentrations, and hence may be effective in reducing cardiovascular risk.

Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (~10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.

Withholding, discontinuing and withdrawing medications in dementia patients at the end of life:a neglected problem in the disadvantaged dying?

Recent years have seen a growing recognition that dementia is a terminal illness and that patients with advanced dementia nearing the end of life do not currently receive adequate palliative care. However, research into palliative care for these patients has thus far been limited. Furthermore, there has been little discussion in the literature regarding medication use in patients with advanced dementia who are nearing the end of life, and discontinuation of medication has not been well studied despite its potential to reduce the burden on the patient and to improve quality of life. There is limited, and sometimes contradictory, evidence available in the literature to guide evidence-based discontinuation of drugs such as acetylcholinesterase inhibitors, antipsychotic agents, HMG-CoA reductase inhibitors (statins), antibacterials, antihypertensives, antihyperglycaemic drugs and anticoagulants. Furthermore, end-of-life care of patients with advanced dementia may be complicated by difficulties in accurately estimating life expectancy, ethical considerations regarding withholding or withdrawing treatment, and the wishes of the patient and/or their family. Significant research must be undertaken in the area of medication discontinuation in patients with advanced dementia nearing the end of life to determine how physicians currently decide whether medications should be discontinued, and also to develop the evidence base and provide guidance on systematic medication discontinuation.

CD11b+ bone marrow-derived monocytes are the major leukocyte subset responsible for retinal capillary leukostasis in experimental diabetes in mouse and express high levels of CCR5 in the circulation

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

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.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

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.

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase

actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al,, Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohesyl)-[H-3]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation, These data strongly suggest that plant ADP is phosphorylation by CDPK(s), a class of protein kinases unique to plants and protozoa. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Treatment approaches for diabetes and dyslipidemia

Dyslipidemia is an important risk factor for cardiovascular complications in persons with diabetes. Low-density lipoprotein-cholesterol (LDL-C) is the 'cornerstone' for assessment of lipoprotein-associated risk. However, LDL-C levels do not reflect the classic 'diabetic dyslipidemia' of hypertriglyceridemia and low high-density lipoprotein-cholesterol (HDL-C). Measurements of plasma apolipoprotein B100 concentrations and non-HDL-C may improve the definition of dyslipidemia. Statins, nicotinic acid and fibrates have roles in treating dyslipidemia in diabetes. Residual risk (i.e. risk that persists after correction of 'conventional' plasma lipoprotein abnormalities) is a new concept in the role of dyslipidemia in the pathogenesis of diabetic vascular complications. For example, regardless of plasma levels, lipoprotein extravasation through a leaking retinal blood barrier and subsequent modification may be crucial in the development of diabetic retinopathy. The current approach to the management of dyslipidemia in diabetes is briefly summarized, followed by a discussion of new concepts of residual risk and emerging lipoprotein-related mechanisms for vascular disease in diabetes.

Revascularization versus medical therapy for renal-artery stenosis

Percutaneous revascularization of the renal arteries improves patency in atherosclerotic renovascular disease, yet evidence of a clinical benefit is limited.