High nitrate levels in skeletal muscle contribute to nitric oxide generation via a nitrate/nitrite reductive pathway in mice that lack the nNOS enzyme.

Introduction: Nitric oxide (NO) is a vasodilator gas that plays a critical role in mitochondrial respiration and skeletal muscle function. NO is endogenously generated by NO synthases: neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), or inducible NO synthase (iNOS). NO in skeletal muscle is partly generated by nNOS, and nNOS deficiency can contribute to muscular dystrophic diseases. However, we and others discovered an alternative nitrate/nitrite reductive pathway for NO generation: nitrate to nitrite to NO. We hypothesized that nitrate supplementation would increase nitrate accumulation in skeletal muscle and promote a nitrate/nitrite reductive pathway for NO production to compensate for the loss of nNOS in skeletal muscle. Methods: Wild-type (WT) and genetic nNOS knockout (nNOS-/-) mice were fed normal chow (386.9 nmol/g nitrate) and subjected to three treatments: high-nitrate water (1 g/L sodium nitrate for 7 days), low-nitrate diet (46.8 nmol/g nitrate for 7 days), and low-nitrate diet followed by high-nitrate water for 7 days each. Results: High-nitrate water supplementation exhibited a greater and more significant increase in nitrate levels in skeletal muscle and blood in nNOS-/- mice than in WT mice. A low-nitrate diet decreased blood nitrate and nitrite levels in both WT and nNOS-/- mice. WT and nNOS-/- mice, treated with low-nitrate diet, followed by high-nitrate water supplementation, showed a significant increase in nitrate levels in skeletal muscle and blood, analogous to the increases observed in nNOS-/- mice supplemented with high-nitrate water. In skeletal muscle of nNOS-/- mice on high-nitrate water supplementation, on low-nitrate diet, and in low-high nitrate treatment, the loss of nNOS resulted in a corresponding increase in the expression of nitrate/nitrite reductive pathway-associated nitrate transporters [sialin and chloride channel 1 (CLC1)] and nitrate/nitrite reductase [xanthine oxidoreductase (XOR)] but did not show a compensatory increase in iNOS or eNOS protein and eNOS activation activity [p-eNOS (Ser1177)]. Discussion: These findings suggest that a greater increase in nitrate levels in skeletal muscle of nNOS-/- mice on nitrate supplementation results from reductive processes to increase NO production with the loss of nNOS in skeletal muscle.

Distribution of dietary nitrate and its metabolites in rat tissues after 15N-labeled nitrate administration.

The reduction pathway of nitrate (NO3-) and nitrite (NO2-) to nitric oxide (NO) contributes to regulating many physiological processes. To examine the rate and extent of dietary nitrate absorption and its reduction to nitrite, we supplemented rat diets with Na15NO3-containing water (1 g/L) and collected plasma, urine and several tissue samples. We found that plasma and urine showed 8.8- and 11.7-fold increases respectively in total nitrate concentrations in 1-day supplementation group compared to control. In tissue samples-gluteus, liver and eyes-we found 1.7-, 2.4- and 4.2-fold increases respectively in 1-day supplementation group. These increases remained similar in 3-day supplementation group. LC-MS/MS analysis showed that the augmented nitrate concentrations were primarily from the exogenously provided 15N-nitrate. Overall nitrite concentrations and percent of 15N-nitrite were also greatly increased in all samples after nitrate supplementation; eye homogenates showed larger increases compared to gluteus and liver. Moreover, genes related to nitrate transport and reduction (Sialin, CLC and XOR) were upregulated after nitrate supplementation for 3 days in muscle (Sialin 2.3-, CLC1 1.3-, CLC3 2.1-, XOR 2.4-fold) and eye (XOR 1.7-fold) homogenates. These results demonstrate that dietary nitrate is quickly absorbed into circulation and tissues, and it can be reduced to nitrite in tissues (and likely to NO) suggesting that nitrate-enriched diets can be an efficient intervention to enhance nitrite and NO bioavailability.

The Proanthocyanidin-Rich Fraction Obtained from Red Rice Germ and Bran Extract Induces HepG2 Hepatocellular Carcinoma Cell Apoptosis.

This study aims to determine the anti-carcinogenic effects of the proanthocyanidin-rich fraction (PRFR) obtained from red rice germ and bran extract on HepG2 cells. The PRFR obtained from red rice germ and bran extract could reduce the cell viability of HepG2 cells as shown by the IC50 value at 20 µg/mL. Notably, PRFR concentrations at 20 and 40 µg/mL significantly increased the number of cells in the G2/M phase from 25.7% ± 1.4%in the control group to 36.2% ± 3.4% (p < 0.01) and 48.9% ± 2.6% (p < 0.0001), respectively, suggesting that the cells were arrested in this phase, which was confirmed by the reduction of survival proteins, including cyclin B1 and cdc25. Moreover, the PRFR at 20 and 40 µg/mL could induce cell death via the apoptosis cascade, indicated by the percentage of total apoptotic cells from 9.9% ± 3.1% in the control group to 41.1 ± 3.9 (p < 0.0001) and 82.2% ± 5.8% (p < 0.0001), respectively. This was clarified by increasing apoptotic proteins (such as cleaved PARP-1, cleaved caspase-8 and cleaved caspase-3) and decreasing anti-apoptotic protein survivin without p53 alterations. These results demonstrated that the PRFR obtained from red rice germ and bran extract could inhibit cell proliferation and induce cell apoptosis in HepG2 cells via survivin, which could potentially serve as a new target for cancer therapeutics making it an excellent "lead candidate" molecule for in vivo proof-of concept studies.

Hepcidin suppression in ß-thalassemia is associated with the down-regulation of atonal homolog 8.

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in ß-thalassemia. We found that inhibition of hepcidin expression in ß-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in ß-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with ß-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the ß-thalassemia patient sera. In conclusion, hepcidin suppression in ß-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in ß-thalassemia.

Iron-Chelating and Anti-Hemolytic Properties of Ethanolic Extract of Lotus (Nelumbonucifera Gaertn) Leaves.

BACKGROUND: Iron overload is the major consequence of blood transfusion in ß-thalassemia patients. Redox iron plays a critical role in the formation of reactive oxygen species and subsequently leads to oxidative stress damage in many cells, especially red blood cells and hepatocytes. Iron deposition in hepatocytes is associated with fibrosis and cirrhosis. Polyphenolic compounds found in natural products are interesting iron chelators and antioxidants. OBJECTIVE: This study aims to evaluate the iron-chelating properties and free-radical scavenging activities of lotus leaf extract in iron-loaded HepG2 cells. MATERIAL AND METHOD: Lotus (Nelumbonucifera Gaertn) leaves were extracted with 80% (v/v) ethanol. The extract was examined for free-radical scavenging activity by using 2, 2-diphenyl-1-picrylhydrazyl assay (DPPH assay); iron-binding and anti-hemolytic activities using spectrophotometrical method. Iron-depriving activity of the extract was determined in iron loaded human hepatocellular (HepG2) cells using fluorescence technique. RESULTS: The lotus extract showed antioxidant and anti-hemolytic activities in a concentration-dependent manner. Furthermore, it was able to bind iron rapidly and was saturated within 10 minute. With 24-hour treatment, this extract dose dependently decreased the level of labile iron pool in iron loaded HepG2 cells. CONCLUSION: Lotus leaf extract had strong antioxidant activities, iron chelating properties on iron loaded HepG2 cells and anti-hemolytic activity.

Identification of dengue virus binding proteins using affinity chromatography.

Several studies have identified putative dengue virus receptors using virus overlay protein binding assays (VOPBA) with some apparent success. Given that this technique relies upon the use of electrophoresis of proteins through polyacrylamide gels with varying amounts of protein denaturation, the physiological relevance of the proteins isolated is open to question. To address this issue a Sepharose 4B-dengue virus serotype 2-affinity column was constructed to selectively bind dengue virus binding proteins from HepG2 (liver) cell membrane preparations. Results show that GRP78, but not the 37/67 kDa high affinity laminin receptor, was specifically bound by the column. This result is consistent with earlier work and shows that while affinity chromatography may provide a useful adjunct to VOPBA based studies particularly in cases where proteins maybe sensitive to denaturation, proteins isolated by VOPBA can be physiologically relevant.

A simplified PCR methodology for semiquantitatively analyzing dengue viruses.

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.