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Pietrzkowski Z, Phelan M, Keller R, Shu C, Argumedo R, Reyes-Izquierdo T. Clin Interv Aging. 2014;9:895-899. It has come to the journal’s and authors’ attention that errors were made during the publication process leading to information being left off the published version that was included in the original drafts. The correct author contribution and funding statements are shown: Author Contributions Zbigniew Pietrzkowski directed the study, Michael Phelan performed all data analysis. Robert Keller organized and helped execute clinical protocols in the study. Cynthia Shu and Ruby Argumedo helped in the data analysis and data compilation. Tania Reyes-Izquierdo led the manuscript writing and final edition and functions as the corresponding author. FundingThe present study was funded by FutureCeuticals, Inc. Further to the above the authors also wish to update the disclosure statement: Disclosure At the time of submission, Dr. Pietrzkowski, Dr. Reyes-Izquierdo, and Dr. Shu were employed by VDF FutureCeuticals, Inc., which funded the study and supplied the calcium fructoborate that was used in the study. Mr. Keller was employed by NutraClinical, Inc.Dr. Pietrzkowski is a named inventor on several patents relating to calcium fructoborate. In 2017, following publication, Dove Medical Press which publishes Clinical Interventions in Aging, became part of the Taylor Francis Group, which is a supporter of the All Trials campaign, an initiative that advocates for the registration of all clinical trials. In furtherance of these goals, the journal has requested that the authors retrospectively register the clinical trial reported in this article. Read the original article

Zbigniew Pietrzkowski,1 Michael J Phelan,2 Robert Keller,3 Cynthia Shu,1 Ruby Argumedo,1 Tania Reyes-Izquierdo11FutureCeuticals, Inc., Applied BioClinical Laboratory; 2Department of Statistics, School of Information and Computer Science, University of California at Irvine; 3NutraClinical Inc., Irvine, CA, USAAbstract: Calcium fructoborate (CFB) at a dose of 110 mg twice per day was previously reported to improve knee discomfort during the first 14 days of treatment. In this study, 60 participants with self-reported knee discomfort were randomized into two groups receiving CFB or placebo. Initial levels of knee discomfort were evaluated by Western Ontario and McMaster Universities Arthritis Index (WOMAC) and McGill Pain Questionnaire (MPQ) scores at the beginning of the study and also at 7 and 14 days after treatment. Results showed that supplementation with CFB significantly improved knee discomfort in the study subjects; significant reductions of mean within-subject change in WOMAC and MPQ scores were observed for the CFB group compared to the placebo group at both 7 and 14 days after treatment. Estimated treatment differences for the MPQ score were -5.8 (P=0.0009) and -8.9 (P

Zbigniew Pietrzkowski,1 Ruby Argumedo,1 Cynthia Shu,1 Boris Nemzer,2 Slawomir Wybraniec,3 Tania Reyes-Izquierdo1 1Applied BioClinical Inc., Irvine, CA, USA; 2FutureCeuticals Inc., Momence, IL, USA; 3Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, Cracow, Poland Aim: We aimed to evaluate the effect of a betalain-rich red beet concentrate (BRC) on joint discomfort and joint function. Methods: Individuals with self-reported knee discomfort were randomized and blinded to treatment with either oral BRC (50 mg twice a day) or placebo. Symptoms of discomfort and joint function were assessed using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and the McGill Pain Questionnaire (MPQ) before treatment and after 5 and 10 days of treatment. The general level of energy was evaluated using a visual analog scale (VAS) at the same time points. Results: In all, 40 individuals were evaluated in this study, 20 in the BRC group and 20 in the placebo group. The mean participant age was 55 years (range: 45–65). All participants completed the study. Individuals receiving BRC had a 27% better MPQ score after 10 days than did individuals in the placebo group (P=0.003). Furthermore, knee function, as measured by WOMAC score, was 26% better in the BRC group than in the placebo group (P

Tania Reyes-Izquierdo,1 Boris Nemzer,2 Ruby Argumedo,1 Cynthia Shu,1 Zb Pietrzkowski1 1Applied BioClinical Inc., Irvine, CA, 2FutureCeuticals Inc, Momence, IL, USA Background: Dermaval™ is a composite formulation of various phytochemical-rich plant materials as quantified using high-performance liquid chromatography. This blend exerts inhibitory activity on human neutrophil elastase (HNE). An acute, crossover clinical study was performed to assess the effects of Dermaval on glucose-induced HNE activity in 20 healthy subjects. Methods: Participants served as their own controls during this 3-day trial. On day 1, all study participants were fasted and given only 300 mL of water. Blood was drawn before treatment and 60 and 120 minutes post treatment. On day 2, participants were fasted, treated with 75 g of glucose, and similarly tested. On day 3, participants consumed a 50 mg serving of Dermaval followed by 75 g of glucose 15 minutes later. HNE concentration and HNE total activity were determined using enzyme-linked immunosorbent assay. Results: Average values for HNE activity in the control group (day 1) did not change. Treatment with a single dose of glucose (day 2) increased blood HNE activity by 175% over baseline levels (P=0.005) during the first 60 minutes. Pretreatment with Dermaval on day 3 prevented the glucose-induced increase in HNE activity (P=0.005 at 60 minutes and P=0.03 at 120 minutes versus respective day 2 values). Interestingly, ingestion of 75 g of glucose resulted in the same blood glucose levels on days 2 and 3, indicating that ingestion of Dermaval did not affect glucose absorption. Conclusion: These results suggest that Dermaval acutely inhibits glucose-induced HNE activity. Further investigations are needed to elucidate the direct and/or indirect mechanism of this effect and to verify whether treatment with Dermaval without glucose may acutely affect activity of HNE as well. Keywords: crossover study, human neutrophil elastase, glucose

Abstract Background Measuring the effects of the acute intake of natural products on human biomarker concentrations, such as those related to oxidation and inflammation, can be an advantageous strategy for early clinical research on an ingredient or product. Methods 31 total healthy subjects were randomized in a double-blinded, placebo-controlled, acute pilot study with post-hoc subgroup analysis on 20 of the subjects. The study examined the effects of a single dose of a polyphenol-rich beverage (PRB), commercially marketed as "SoZo®", on serum anti-inflammatory and antioxidant markers. In addition, phytochemical analyses of PRB, and in vitro antioxidant capacity were also performed. Results At 1 hour post-intake, serum values for 8-iso-PGF2-alpha and advanced oxidation protein products decreased significantly by 40% and 39%, respectively. Additionally, there was a trend toward decreased C-reactive protein, and increased nitric oxide levels. Both placebo and PRB treatment resulted in statistically significant increases in hydroxyl radical antioxidant capacity (HORAC) compared to baseline; PRB showed a higher percent change (55-75% versus 23-74% in placebo group), but the two groups did not differ significantly from each other. Conclusions PRB produced statistically significant changes in several blood biomarkers related to antioxidant/anti-inflammatory effects. Future studies are justified to verify results and test for cumulative effects of repeated intakes of PRB. The study demonstrates the potential utility of acute biomarker measurements for evaluating antioxidant/anti-inflammatory effects of natural products.

Human peripheral blood mononuclear cells (PBMC) were used as a biological model to measure intracellular ATP (iA TP) reactive oxygen species (ROS), Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR), in response to acute ex vivo treatments with HH2o. "HH2o ", a fermented, food-based material commercially marketed as "Mitochroma™" has been identified as a potential modulator of iATP, OCR and ROS in these cells. PBMC were exposed to HH2o (0.1, 0.01 and 0.001% w/v) and Carbonyl-cyanide p-trifluoro-methoxy-phenyl-hydrazone (FCCP) or Oligomycin, two known modulators of mitochondrial activity were used as controls. Results showed that treatment ofthe cells with HH20 resulted in increase of iATP up to 220% and reduction of iADP by up to 40% while iROS levels remained unchanged. At the same time, OCR level was increased up to 24% compared to initial baseline. These results suggest that HH2o acts similarly to uncoupling protein 2 (UCP2) when induced and that it may have potential use in developing health-promoting products. These results support the use of freshly isolated human PBMCs as an experimental ex vivo model to detect the effects of nutraceutical products on dynamic cell metabolites, such as oxygen consumption, ATP, ADP and ROS. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the possible correlation between the bioactivity and the phytochemical profile of four betalain-rich extracts from Portulaca grandiflora Hook. The HPLC-DAD-ESI-MS analysis indicated the presence of 19 betaxanthins and two betacyanins. The highest concentrations of betaxanthins (982 mg/100 g DE) and betacyanins (650 mg/100 g DE) were noticed in orange and purple flowers extracts, respectively. The HPLC-DAD-ESI-HRMS/MS analyses revealed the presence of a total of 71 compounds. Fifteen new betaxanthins and fifty other metabolites were identified for the first time. The antioxidant activity of the studied flower extracts increased in the sequence of yellow < orange < purple < red (0.066−0.176 mM TE/g DE). Betalains showed less effect on the antioxidant activity of extracts than other metabolites did. Extracts from yellow and orange flowers were more active against Gram-positive bacteria (MIC = 4−16 mg/L), whereas extracts from red and purple flowers were slightly more active against Gram-negative bacteria (MIC = 16−32 mg/L). All the extracts showed the same activity against yeasts (MIC = 32 mg/L). Betaxanthins were active against Gram-positive bacteria, whereas betacyanins were active against Gram-negative bacteria. Remaining metabolites also exhibited antimicrobial activities. The cytotoxicity assessment showed that the P. grandiflora extracts were non-toxic to normal VERO cells. No significant antiviral activity towards Human Herpesvirus type 1 was observed (62 µg/mL). Among the tested varieties, the purple one showed anticancer selectivity towards colon carcinoma cells (RKO).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot ( Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.

Neutrophils produce hypochlorous acid (HOCl) as well as other reactive oxygen species as part of a natural innate immune response in the human body; however, excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. Comparison of the in vitro anti-hypochlorite activities of a novel betalain-rich red beetroot ( Beta vulgaris L.) extract with its pure betalainic pigments revealed that the extract had the highest anti-hypochlorite activity, far exceeding the activity of all of the betalainic derivatives and selected reference antioxidants. This suggests that it may be an important food-based candidate for management of inflammatory conditions induced by excessive HOCl production. Among all pigments studied, betanidin exhibited the highest activity across the pH range.

Hypochlorous acid (HOCl) produced by neutrophils is a part of the natural innate immune response system in the human body, but excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. In this contribution, chlorination mechanism and position of the electrophilic substitution in betacyanins was studied by high-resolution mass spectrometry and further structural analyses by NMR techniques, which completed the identification of the chlorinated betacyanins. For the study on the influence of the position of decarboxylation on the chlorination mechanism, a comparison of the chlorination position between betanin as well as 17-, and 2,17-decarboxylated betanins was performed. The structural study confirmed that the chlorination position in betanin occurs within the dihydropyridinic moiety at carbon C-18. Therefore, out of the aqueous free chlorine equilibrium species: HOCl, OCl - , Cl 2 , and Cl 2 O, the most potent chlorinating agents are HOCl and Cl 2 O postulated previously and the attack of the Cl ⁺ ion on the carbon C-18 with a cyclic intermediate version is considered., Competing Interests: The authors declare no conflict of interest.

The use of natural pigments such as betalains in food and health-related products is often limited by said pigments' relative oxidative stabilities in the products or physiological matrices. Determination of the mechanism of oxidation may inform future development and delivery of better stabilized molecules for improved outcomes. In order to best determine the oxidation mechanism of betanin, a natural food colorant, our efforts were directed toward structural elucidation (LCMS-IT-TOF and NMR) of previously tentatively identified key dehydrogenation products that had been generated as a result of betanin, decarboxylated betanin, and neobetanin oxidation by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radicals. The resultant oxidation products, the neo-derivatives, were the most stable and survived the preparative isolation and purification process. Structural analyses subsequently confirmed that these compounds, as well as neobetanin, were also the key products of alternative pathways of betanin and 2-decarboxy-betanin oxidation when catalyzed by Cu 2+ cations in aqueous solutions at pH close to neutral. Therefore, the structures of the following five neo- or xanneo-derivatives (14,15- or 2,3,14,15-dehydrogenated derivatives, respectively) were confirmed: neobetanin, 2-decarboxy-neobetanin, 2-decarboxy-xanneobetanin, 2,17-bidecarboxy-xanneobetanin, and 2,15,17-tridecarboxy-xanneobetanin. This research confirmed that Cu 2+ -catalyzed oxidation of betanin and 2-decarboxy-betanin results in generation of neo-derivatives of betanin. In contrast, Cu 2+ -catalyzed oxidation of 17-decarboxy-betanin and 2,17-bidecarboxy-betanin resulted mostly in formation of betanin xan-derivatives. A relevant mechanism of Cu 2+ -catalyzed oxidation of the pigments is proposed herein that suggests that the oxidation of betanin can possibly occur in the region of the dihydropyridinic ring and can omit the stage of methide quinone formation in the dihydroindolic system.

A comparative chromatographic evaluation of chlorinated decarboxylated betanins and betanidins generated under activity of hypochlorous acid exerted upon these highly antioxidative potent decarboxylated pigments derived from natural sources was performed by LC-DAD-ESI-MS/MS. Comparison of the chromatographic profiles of the chlorinated pigments revealed two different directions of retention changes in relation to the corresponding substrates. Chlorination of all betacyanins that are decarboxylated at carbon C-17 results in an increase of their retention times. In contrast, all other pigments (the non-decarboxylated betacyanins as well as 2-decarboxy- and 15-decarboxy-derivatives) exhibit lower retention after chlorination. During further chromatographic experiments based upon chemical transformation of the related pigments (decarboxylation and deglucosylation), the compounds' structures were confirmed. The elaborated method for determination of chlorinated pigments enabled analysis of a chlorinated red beet root extract that was submitted to the MPO/H 2 O 2 /Cl - system acting under inflammation-like conditions (pH 5). This indicates a promising possibility for measurement of these chlorinated pigments as indicators of specific inflammatory states wherein betacyanins and decarboxylated betacyanins act as hypochlorite scavengers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Increased cellular ATP levels have the potential to enhance athletic performance. A proprietary blend of ancient peat and apple extracts has been supposed to increase ATP production. Therefore, the purpose of this investigation was to determine the effects of this supplement on athletic performance when used during 12 weeks of supervised, periodized resistance training., Methods: Twenty-five healthy, resistance-trained, male subjects completed this study. Subjects supplemented once daily with either 1 serving (150 mg) of a proprietary blend of ancient peat and apple extract (TRT) or an equal-volume, visually-identical placebo (PLA) daily. Supervised resistance training consisted of 8 weeks of daily undulating periodized training followed by a 2 week overreach and a 2 week taper phase. Strength was determined using 1-repetition-maximum (1RM) testing in the barbell back squat, bench press (BP), and deadlift exercises. Peak power and peak velocity were determined during BP at 30 % 1RM and vertical jump tests as well as a 30s Wingate test, which also provided relative power (watt:mass), Results: A group x time interaction was present for squat 1RM, deadlift 1RM, and vertical jump peak power and peak velocity. Squat and deadlift 1RM increased in TRT versus PLA from pre to post. Vertical jump peak velocity increased in TRT versus PLA from pre to week 10 as did vertical jump peak power, which also increased from pre to post. Wingate peak power and watt:mass tended to favor TRT., Conclusions: Supplementing with ancient peat and apple extract while participating in periodized resistance training may enhance performance adaptations., Trial Registration: ClinicalTrials.gov registration ID: NCT02819219 , retrospectively registered on 6/29/2016.

This study presents a comparative evaluation of chlorination of betanin, betanidin, and neobetanin exposed to sodium hypochlorite and myeloperoxidase (MPO)/H2O2/Cl(-) systems. For betanin/betanidin, the chlorination takes place at the aglycone unit, but for neobetanin, no chlorinated products in the reaction mixtures can be detected. In the RP-HPLC system, monochloro-betanin/-betanidin were eluted earlier than their corresponding nonchlorinated substrates. An influence of Cl(-) concentration on betanin/betanidin chlorination efficiency in sodium hypochlorite and MPO systems was investigated. At pH 3-5, the yields of formed monochloro-betanin/-betanidin decrease dramatically at higher Cl(-) concentrations, indicating that generated Cl2 is not the chlorinating agent in the presence of sodium hypochlorite. The intriguing low activity of Cl2 in betanin/betanidin chlorination compared to HOCl and/or Cl2O can be explained by a special position of the attack by molecules of HOCl and/or Cl2O. In the MPO/H2O2/Cl(-) system, the highest efficiency of monochloro-betanin/-betanidin generation is observed at pH 5.

Calcium fructoborate (CFB) has been reported as supporting healthy inflammatory response. In this study, we assess the effects of CFB on blood parameters and proinflammatory cytokines in healthy subjects. This was a randomized, double-blinded, placebo-controlled trial. Participants received placebo or CFB at a dose of 112 mg/day (CFB-1) or 56 mg/day (CFB-2) for 30 days. Glucose, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG), C-reactive protein (CRP), homocysteine, interleukin 1 beta (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP-1) were determined before and after supplementation. CFB-1 showed a reduction in blood levels of CRP by 31.3 % compared to baseline. CFB-1 and CFB-2 reduced LDL levels by 9.8 and 9.4 %, respectively. CFB-1 decreased blood homocysteine by 5.5 % compared with baseline, whereas CFB-2 did not have a significant effect. Blood levels of TG were reduced by 9.1 and 8.8 % for CFB-1 and CFB-2, respectively. Use of both CFB-1 and CFB-2 resulted in significantly reduced IL-6 levels, when compared within and between groups. IL-1β was reduced by 29.2 % in the CFB-1 group. Finally, CFB-1 and CFB-2 reduced MCP-1 by 31 and 26 %, respectively. Our data indicate that 30-day supplementation with 112 mg/day CFB (CFB-1) resulted in a significant reduction of LDL, TG, TC, IL-1β, IL-6, MCP-1, and CRP. HDL levels were increased, when compared to baseline and placebo. These results suggest that CFB might provide beneficial support to healthy cardiovascular systems by positively affecting these blood markers (ClinicalTrials.gov, ISRCTN90543844; May 24, 2012 ( http://www.controlled-trials.com/ISRCTN90543844 )).

ORAC and other in vitro methods have to date proved useful in measuring antioxidant potential in foods. In order to better understand the potential relationship between diet and free radical production/mitigation, an in vivo analytic method can provide new insight into directly measuring reactive oxidant species (ROS). Dihydrorhodamine-6G (DHR6G) is indiscriminate to the various free radicals found in humans, and therefore can be useful in quantifying total ROS in vivo. Our aim was to investigate whether the total ROS in human subjects can be quantified using DHR6G after intake of a blend of antioxidants-rich fruit and vegetable-based materials. Twelve participants were given 100 mg of a proprietary blend of fruit, vegetable, and herb powders and concentrates commercially marketed under the trade name "Spectra™". Blood samples were collected at 0, 60, 120 and 180 min and were subsequently tested for ROS in serum using DHR6G as a fluorescent probe. By quantifying this fluorescence, we were able to measure ROS concentrations in human blood. This method is both reliable and efficient for evaluating the efficacy of antioxidants against ROS in vivo. Our data indicate that eleven participants responded to the intake of Spectra™ by significant decreases of ROS concentrations.

The present single-dose study was performed to assess the effect of whole coffee fruit concentrate powder (WCFC), green coffee caffeine powder (N677), grape seed extract powder (N31) and green coffee bean extract powder (N625) on blood levels of brain-derived neurotrophic factor (BDNF). Randomly assorted groups of fasted subjects consumed a single, 100mg dose of each material. Plasma samples were collected at time zero (T0) and at 30 min intervals afterwards, up to 120 min. A total of two control groups were included: subjects treated with silica dioxide (as placebo) or with no treatment. The collected data revealed that treatments with N31 and N677 increased levels of plasma BDNF by about 31% under these experimental conditions, whereas treatment with WCFC increased it by 143% (n 10), compared with baseline. These results indicate that WCFC could be used for modulation of BDNF-dependent health conditions. However, larger clinical studies are needed to support this possibility.

A comprehensive nonenzymatic oxidation mechanism in betanin plant pigment as well as its derivatives, 2-decarboxybetanin, 17-decarboxybetanin, 2,17-bidecarboxybetanin, and neobetanin, in the presence of ABTS cation radicals was investigated by LC-DAD-ESI-MS/MS. The main compounds formed during the first step of betanin and 2-decarboxybetanin oxidation are 2-decarboxy-2,3-dehydrobetanin and 2-decarboxyneobetanin, respectively. In contrast to betanin, the reaction mechanism for 2-decarboxybetanin includes more oxidation pathways. Parallel transformation of 2-decarboxybetanin quinone methide produces neoderivatives according to an alternative reaction that omits the presumably more stabile intermediate 2-decarboxy-2,3-dehydrobetanin. The main oxidation product after the first reaction step for both 17-decarboxybetanin and 2,17-bidecarboxybetanin is 2,17-decarboxy-2,3-dehydrobetanin. This product is formed through irreversible decarboxylation of the 17-decarboxybetanin quinone methide or by oxidation of 2,17-bidecarboxybetanin. Oxidation of neobetanin results primarily in a formation of 2-decarboxy-2,3-dehydroneobetanin by a decarboxylative transformation of the formed neobetanin quinone methide. The elucidated reaction scheme will be useful in interpretation of redox activities of betalains in biological tissues and food preparations.

CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

The antioxidative mechanism of action of betalains is of significant interest because these pigments are recently emerging as highly bio-active natural compounds with potential benefits to human health. Betanidin, the basic betacyanin, comprises the 5,6-dihydroxyl moiety, which results in its high antioxidant activity. Oxidation of betanidin by voltammetric techniques and chro matographic identification of the oxidation products with spectrophotometric and mass spectrometric detection (LC-DAD-MS/MS) were performed. Two main oxidation peaks for betanidin are observable at pH 3-5. These peaks become merged at higher pH, suggesting a different mechanism of oxidation at higher and lower pH values. The low oxidation potential of betanidin confirms its very strong reduction properties. The presence of two prominent oxidized products, 2-decarboxy-2,3-dehydrobetanidin and 2,17-bidecarboxy-2,3-dehydrobetanidin, indicates their generation through two reaction routes with two different quinonoid intermediates: dopachrome derivative and quinone methide. Both lead to the decarboxylative dehydrogenation of betanidin. Subsequent oxidation and rearrangement of the conjugated chromophoric system results in formation of 14,15-dehydrogenated derivatives.

Background: Measuring the effects of the acute intake of natural products on human biomarker concentrations, such as those related to oxidation and inflammation, can be an advantageous strategy for early clinical research on an ingredient or product., Methods: 31 total healthy subjects were randomized in a double-blinded, placebo-controlled, acute pilot study with post-hoc subgroup analysis on 20 of the subjects. The study examined the effects of a single dose of a polyphenol-rich beverage (PRB), commercially marketed as "SoZo(®)", on serum anti-inflammatory and antioxidant markers. In addition, phytochemical analyses of PRB, and in vitro antioxidant capacity were also performed., Results: At 1 hour post-intake, serum values for 8-iso-PGF2-alpha and advanced oxidation protein products decreased significantly by 40% and 39%, respectively. Additionally, there was a trend toward decreased C-reactive protein, and increased nitric oxide levels. Both placebo and PRB treatment resulted in statistically significant increases in hydroxyl radical antioxidant capacity (HORAC) compared to baseline; PRB showed a higher percent change (55-75% versus 23-74% in placebo group), but the two groups did not differ significantly from each other., Conclusions: PRB produced statistically significant changes in several blood biomarkers related to antioxidant/anti-inflammatory effects. Future studies are justified to verify results and test for cumulative effects of repeated intakes of PRB. The study demonstrates the potential utility of acute biomarker measurements for evaluating antioxidant/anti-inflammatory effects of natural products.

Interacting melanocytes and keratinocytes in the epidermis create a unique structure in which keratinocytes regulate the growth of melanocytes and the expression of cell surface molecules. The critical role of communication between these cells means that any anti-melanoma drug should be studied in the context of its possible influence on keratinocytes. For that reason, this study focused on comparing the influence of daunomycin on human melanoma cells and on keratinocytes in vitro. The effects were studied by cytochemical methods (TUNEL, FITC-Annexin V labelling, endocytosis activity assay, measurements of DNA content) and morphological methods (measurements of cell surface area, perimeter, extension, dispersion and elongation) to verify the hypothesis of differential response. The results of our research demonstrate that keratinocytes are less susceptible than melanoma cells to daunomycin treatment in vitro. Keratinocytes are also able to resume growth when the drug is removed from the medium, whereas melanoma cells have not demonstrated this capacity. Apoptosis was identified as the mechanism by which the drug exerts its cytostatic effects.

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, alpha-chemokine stromal-derived factor 1 (SDF-1), is secreted in muscle tissue. These cell lines responded to SDF-1 stimulation by chemotaxis, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT serine-threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34(+) hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4(+) satellite cells just as they chemoattract CD34(+) cells. Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34(+) cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle- or bone marrow-derived fibroblasts. Based on these observations, we postulate that the SDF-1-CXCR4 axis is involved in chemoattracting circulating CXCR4(+) muscle stem/progenitor and circulating CXCR4(+) hematopoietic CD34(+) cells to both muscle and bone marrow tissues. Thus, it appears that tissue-specific stem cells circulating in peripheral blood could compete for SDF-1(+) niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.

CD44H is a transmembrane protein involved in cell-cell and cell-matrix interactions. In melanoma cells CD44H influences motility and invasiveness. Daunomycin (daunorubicin) is an anthracycline antibiotic, capable to inducing apoptosis in many cell lines. The data presented in this report show that 12-hours treatment with clinically applied concentration of daunomycin led to apoptotic death of a portion of the investigated melanoma cell population. Surviving cells showed random distribution of CD44H and decreased expression of the protein on the cell surface, but no cell blebbing or changes in nuclei. Hypothetical mechanisms concerning a role of CD44H in induction of apoptosis are discussed.

The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34(+) cells and ex vivo expanded early colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst forming unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte (CFU-Meg)-derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor-2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor-alpha, Fas ligand, interferon alpha, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-3 [MCP-3], MCP-4, IL-8, interferon-inducible protein-10, macrophage-derived chemokine [MDC], and platelet factor-4 [PF-4]) to be expressed by CD34(+) cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor-beta1 (TGF-beta1), TGF-beta2, RANTES, MIP-1alpha, MIP-1beta, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34(+) cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34(+) cells; chemo-attract CFU-GM- and CFU-Meg-derived cells as well as other CD34(+) cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM-, CFU-Meg-, and BFU-E-derived cells. It is concluded that normal human CD34(+) cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.

4-Amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was recently synthesized and evaluated in our laboratories for anticancer activities. This compound showed potent in vitro inhibitory effects on the growth of HTB-81 prostate cancer cells and Daudi-lymphoma. In vivo studies showed that the compound could inhibit HTB-81 tumor growth in syngeneic mice by 93% at a daily dose of 8.5 mg/kg for 10 days.

Compounds that could block tumor angiogenesis and induce tumor cell differentiation in malignant gliomas represent a very valuable tool in anticancer treatments. In this paper, we demonstrate that more selective drugs, which interfere with specific cellular targets, could treat glioma more effectively. 8-Cl-cAMP and tiazofurin (TR) are site-specific analogs that selectively inhibit PKAI and IMP dehydrogenase, are directly involved in cell proliferation and apoptosis, and mediate the mitogenic effects of different oncogenes and growth factors. In this study, we have examined influence of 8-Cl-cAMP and TR on the production of an angiogenic factor [vascular endothelial growth factor (VEGF)] by human glioblastoma U251 MG cells, as well as their influence on the expression of a differentiating marker [glial fibrillary acidic protein (GFAP)]. Using a cell proliferation assay, VEGF enzyme-linked immunoassay and GFAP immunocytochemistry we demonstrated the effects of these compounds. Our results demonstrate that 8-Cl-cAMP and TR decrease VEGF production by U251 MG cells, and that under the influence of both agents these cells increase GFAP expression and change their morphology, becoming more differentiated. These findings also suggest that 8-Cl-cAMP and TR may have potential for further investigation of their antiangiogenic and differentiational role in malignant disease such as human gliomas.

CD34(+) cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34(+) cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34(+) cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34(+) cells and CD34(+)KIT(+) cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1alpha, and MIP-1beta and that IFN-gamma stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34(+)KIT(+) cells, even by RT-PCR. Conditioned media from CD34(+) cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34(+) cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34(+) cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.

We have previously reported oligodeoxynucleotides (ODNs) containing 4'-C- and 5'-C-substituted thymidines, which demonstrated certain favorable biophysical and biochemical properties. In this communication, the hybridization and nuclease stability data of the ODNs along with their capability to induce RNase H activity are presented.

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk-1/KDR and Flt-1 respectively. Flk-1/KDR-deficient murine embryos manifest failure of blood-island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt-1 and Flk-1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt-1 and Flk-1/KDR receptors on stem cell rich CD34+ c-kit-R+ Rh123(low) cells. Further studies revealed that Fit-1 R mRNA, but not Flk-1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co-stimulated HEL cell proliferation as well as CFU-GM colony formation from approximately 15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.

We examined the importance of IL-8 receptor B mRNA expression in the growth of non-small cell lung cancer (NSCLC). Using antisense oligonucleotide ICN 197, we were able to inhibit IL-8 R B mRNA expression in vitro. The sequence specific effect of antisense oligonucleotide and down-regulation of IL-8 R B mRNA was shown by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Southern blot analysis. The proliferation of treated cells was measured by 3H thymidine incorporation. We found that treatment of NSCLC cells caused reversible growth inhibition and reversible down regulation of IL-8 R B mRNA. Furthermore, we observed that the treatment of nude mice with oligonucleotide ICN 197 inhibited the growth of tumors developed from NSCLC cells injected subcutaneously. Our data in vitro suggest that IL-8 receptor B mRNA expression is required to maintain the proliferative rate of NSCLC. Based on the data in vivo. oligonucleotide ICN 197 may be considered for the development of novel therapeutic treatment for lung cancer.

The results of transplantation of free autologous fat obtained by blunt syringe suction lipectomy are unpredictable. We examined if adipose tissue viability is compromised by using syringe suction lipectomy and by infiltration of the tissue with local anesthetics. As reference, we used adipose tissue samples excised during elective surgery. Fat obtained intraoperatively and by lipectomy was digested with collagenase to isolate adipocytes. The mechanical damage associated with sample handling and cell isolation in both procedures was similar and did not exceed 6% of the total cell mass. In addition, cells isolated from intraoperative and lipectomy samples did not differ functionally, responded similarly to insulin stimulation of glucose transport and epinephrine-stimulated lipolysis, and retained the same growth pattern in culture. Since during fat transplantation the graft is exposed to local anesthetics at both the donor and the recipient sites, we reexamined adipocyte function in the presence of lidocaine. Lidocaine potently inhibited glucose transport and lipolysis in adipocytes and their growth in culture. That effect, however, persisted only as long as lidocaine was present; after washing, the cells were able to fully regain their function and growth regardless of whether the exposure was as short as 30 minutes or as long as 10 days. These results indicate that adipose tissue obtained by syringe lipectomy consists of fully viable and functional adipocytes, but local anesthetics may halt their metabolism and growth.

The objectives of this study were to develop a new technique to safety, reliably, and in a cosmetically acceptable way, obtain more than 5.0 g of abdominal subcutaneous fat in out-patients, and to investigate whether inhibitory effects of a local anaesthetic on adipose tissue function in vitro are sufficient argument against the use of infiltrative local anaesthesia during fat biopsy to obtain samples for metabolic studies. Measurements were obtained to compare glucose transport and lipolysis response to insulin in adipocytes isolated from subcutaneous abdominal fat obtained: (i) during elective surgery in eight women and four men (BMI 25.8 +/- 3.1 kg/m2); and (ii) from five male and three female out-patients (BMI 25.8 +/- 3.1 kg/m2) by the described novel technique performed under local anaesthesia with Lidocaine. The effects of acute and 11-day exposure to Lidocaine in vitro on adipocyte lipolysis and glucose transport response to insulin, and the growth potential were determined. In vivo exposure of the tissue samples to local anaesthetic by the novel technique had no apparent effect on isolated adipocyte responses to insulin by stimulation of glucose transport or by inhibitor- or adrenaline-stimulated lipolysis; the results were not different to those for adipocytes isolated from fat obtained during elective abdominal surgeries. Lidocaine added in vitro potently inhibited glucose transport and lipolysis in adipocytes, and cell attachment and growth in primary 'ceiling' culture; this effect persisted only as long as Lidocaine was present. After washing, adipocytes fully regained their function and growth regardless of the exposure period, as short as 30 min or as long as 11 days.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated three prostatic cancer cell lines, PC-3, DU-145, and LNCa.FGC, and found that all three cell lines can grow in serum-free medium without the addition of exogenous growth factors. All three cell lines produce substantial amounts of insulin-like growth factor 1 (IGF-1) that is secreted in the medium and they all display constitutively autophosphorylated IGF-1 receptors; two of the cell lines overexpress IGF-1 receptor RNA. The growth of all three cell lines is inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA or by peptide analogues of IGF-1 that compete with IGF-1 binding to its receptor. Our results indicate that these three cell lines grow by an autocrine loop in which the overproduced IGF-1 activates its receptor. Interference with the activation of the receptor leads to cessation of growth.