[Show abstract][Hide abstract] ABSTRACT: A homodimeric, growth-related and time-keeping hydroquinone oxidase (ENOX1) of the eukaryotic cell surface capable of oxidizing intracellular NADH exhibits properties of the ultradian driver of the biological 24 h circadian clock by exhibiting a complex 2 + 3 set of oscillations of copper salts and appear to derive from periodic variations in the ratio of ortho and para nuclear spins of the paired hydrogen atoms of the elongated octahedral structure of the ENOX1 protein bound copper II hexahydrates. A corollary of these observations is that the ortho/para oscillations must occur in a highly synchronized matter. Our findings suggest that water molecules communicate with each other via very low frequency electromagnetic fields and that these fields also appear to be generated by the energetics of the synchronous ortho to para interconversions of the nuclear spin pairs of the water hydrogens. Further evidence for energy absorbed and emitted by water and correlated with ortho/para oscillations of ortho/para spin pairs of water hydrogens is indicated from the auto-oscillations in water luminescence. The emissions oscillate with period lengths of 18.8 min that agree with our previously found period of oscillation of about 18 min for pure water, reflective of ortho to para spin isomers based on measurements of redox potential. The period length of pure water (increased by about 25% in D2O) and varies depending on the dominant cation present (copper salts in solution are unique in that the period length is exactly 24 min). Synchrony is maintained through generation of and response to LFEMF generated by the ortho-para spin pairs. Changes in redox potential sufficient to catalyze NADH oxidation were used to monitor synchronous water oscillations that appear to extend indefinitely over great distances in contiguous bodies of either still or flowing water. Adjacent out-of-phase water samples contained in thin plastic cuvettes auto-synchronize in a matter of seconds when placed side by side. Potential applications from water treatment along with opportunity related to human health ard and maintained, and to be aided by methodological advances in measurement ane anticipated to derive from a better understanding of how water synchrony is generated analysis.
Water 05/2015; 7(5):2082-2100. DOI:10.3390/w7052082 · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze
oxidation of both NADH and hydroquinones as well as carry out protein disulfide-thiol interchange.
They exhibit both prion-like and time-keeping (clock) properties. The oxidative and
interchange activities alternate to generate a regular period of 24 min in length. Here we report
the cloning, expression, and characterization of a plant candidate constitutive ENOX (CNOX or
ENOX1) protein from Arabidopsis lyrata. The gene encoding the 335 (165) amino acid protein is
found in accession XP-002882467. Functional motifs characteristics of ENOX proteins previously
identified by site-directed mutagenesis and present in the candidate ENOX1 protein from plants
include adenine nucleotide and copper binding motifs along with essential cysteines. However, the
drug binding motif (EEMTE) sequence of human ENOX2 is absent. The activities of the recombinant
protein expressed in E. coli were unaffected by capsaicin, EGCg, and other ENOX2-inhibiting
substances. Periodic oxidative activity was exhibited both with NAD(P)H and reduced coenzyme Q
as substrate. Bound copper was necessary for activity and activity was inhibited by the ENOX1-
specific inhibitor simalikalactone D. Addition of melatonin phased the 24-min period such that the
next complete period began 24 min after the melatonin addition as appeared to be characteristic
of ENOX1 activities in general. Periodic protein disulfide-thiol interchange activity also was demonstrated
along with the 2 oxidative plus 3 interchange activity pattern characteristics of the 24-
min ENOX1 protein period. Concentrated solutions of the purified plant ENOX1 protein formed
insoluble aggregates, devoid of enzymatic activity, resembling amyloid. Activity was restored to aggregate preparations by isoelectric focusing.
[Show abstract][Hide abstract] ABSTRACT: ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of
both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prionlike
and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate
a regular period of 24 min in length. Here we report the cloning, expression and characterization
of a constitutive plant ENOX protein activated by both natural (Indole-3-acetic acid, IAA)
and synthetic (2,4-dichlorophenoxyacetic acid, 2,4-D) auxin plant growth regulators with an optimum
of about 1 μM, higher concentrations being less effective. The gene encoding the 213 amino
acid protein (ABP20) is found in EMBL accession number U81162. Functional motifs characteristic
of ENOX1 proteins, previously identified by site-directed mutagenesis, are present in the candidate
auxin-activated ENOX (dNOX, ENOX5), including adenine nucleotide and copper binding motifs
along with essential cysteines and a motif having homology with a previously identified auxinbinding
motif. Periodicity was exhibited by both the oxidative and protein disulfide-thiol interchange
activities as is characteristic for other ENOX proteins. Activity was blocked by the ENOX2-
specific quassinoid inhibitor glaucarubolone and other ENOX2 inhibitors but not by the ENOX1-
specific quassinoid inhibitor simalikalactone D. Activity required both auxin and bound copper.
The inactive auxin 2,3-D was without effects.
Advances in Biological Chemistry 12/2014; 4(07):415-427. DOI:10.4236/abc.2014.47047
[Show abstract][Hide abstract] ABSTRACT: ME-143 (NV-143), a synthetic isoflavone under clinical evaluation for efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol
interchange activity designated ENOX2 (tNOX) and inhibited the growth of cultured cancer cells with EC50s in the range of 20‐50 nM. Purified recombinant ENOX2 also bound ME-143 with a K
d of 43 (40‐50) nM. Both the oxidative and protein disulfide-thiol
interchange activities of ENOX proteins that alternate to generate a complex set of oscillations with a period length of 22 min compared to 24 min for the constitutive counterpart ENOX1 (CNOX) that characterizes ENOX proteins responded to ME-143. Oxidation of NADH or reduced coenzyme Q10
was rapidly blocked. In contrast, the protein disulfide-thiol interchange activity measured from the cleavage of dithiodipyridine (EC50 of ca. 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled
the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ENOX1 (CNOX) forms of either cancer or noncancer cells were unaffected by ME-143 over the range of concentrations inhibiting ENOX2. Taken together,
the findings show that ME-143 binds to ENOX2 with an affinity 4 to 10 times greater than that reported previously for the related anticancer isoflavone, phenoxodiol.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics 10/2014; 22(1). DOI:10.3727/096504014X14077751730270 · 1.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Age-related NADH oxidase (arNOX), a cell surface-located hydroquinone oxidase capable of superoxide generation, appears at age 30 and increases with age thereafter. The ectodomain of arNOX is shed from the cell surface into body fluids including sera and saliva where its activity was measured spectrophotometrically using a reduction of ferricytochrome c as a measure of superoxide generation. The autofluorescence of advanced glycation end products correlates with epidermal arNOX activity as well. To demonstrate protein cross-linking, a fluorescence-labeled analog of tyrosine, tyramine, that would react with proteins carrying arNOX-generated tyrosyl radicals was used. The assay demonstrated the potential for arNOX-induced oxidative damage (dityrosine formation) to human collagen and elastin and to other surface proteins of intact human embryo fibroblasts and frozen sections from epidermal punch biopsies. The findings support a role for arNOX as a major source of oxidative damage leading to cross-linking of skin proteins.
Archives for Dermatological Research 06/2014; 306(7). DOI:10.1007/s00403-014-1472-8 · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Experts agree that one of the more promising strategies in cancer management is early detection coupled with early intervention. In this study, we evaluated an early cancer detection strategy of cancer presence based on serum levels of the cancer-specific transcript variants of ENOX2 in serum coupled with an ENOX2-targeted nutraceutical preparation of green tea concentrate plus Capsicum (Capsol-T(R)) as a strategy of Curative Prevention(R) involving early detection coupled with early intervention in early stage cancer when in its most susceptible and manageable stages.Experimental design: One hundred ten (110) subjects were tested for cancer presence using the ONCOblot(R) Tissue of Origin 2-D gel/western blot protocol for detection of serum presence of transcription variants of the ENOX2 protein. Subjects testing positive for ENOX2 received 350 mg of Capsol-T(R) in capsule form every 4 h including during the night for periods of at least 3 to 6 months or longer after which they were again tested for ENOX2 presence using the ONCOblot(R) Tissue of Origin Cancer Test protocol.
Of the 110 subjects, both male and female, ages 40 to 84, with no evidence of clinical symptoms of cancer, 40% were positive for ENOX2 presence in the ONCOblot(R) Tissue of Origin Cancer Test. After completion of 3 to 17 months of Capsol-T(R) use, 94% of subjects subsequently tested negative for ENOX2 presence.
Oral Capsol-T(R) is well tolerated and, for ENOX2 presence in serum in the absence of clinical cancer symptoms, is consistently effective in reducing the serum ENOX2 levels to below detectable limits.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Reactive oxygen species that are produced by aerobic metabolism and signaling cascades have the potential to play important roles in maintaining homeostatic redox and cell proliferation. When the balance between the production and elimination of reactive oxygen species is perturbed toward production, the result is oxidative stress. High levels of oxidative stress are a general characteristic of cancer. The altered redox state within a tumor microenvironment confers a growth advantage through increased proliferation rates, evasion of apoptosis, and increased resistance to therapeutic compounds. We have tested a synergistic combination of green tea-Camellia sinensis-concentrate and powdered Capsicum powder (TeaFense™/Capsol-T™) as a dietary supplement to reduce oxidative stress as an approach to elimination of malignant cells. Here, we demonstrate that the green tea-powdered Capsicum mixture effectively reduces levels of oxidative stress in both cancer (HeLa) and noncancer (MCF-10A) cells as determined from measurements of levels of the oxidative stress indicator Nrf-2 by western blot analysis. Nrf-2 is a transcription factor that controls an antioxidant response element. Increased expression of Nrf-2 is linked to high levels of oxidative stress and vice versa. Based on levels of Nrf-2, the mixture of green tea concentrate plus powdered Capsicum reduced oxidative stress by more than 50% compared with 15% by the green tea concentrate alone.
Journal of Dietary Supplements 12/2013; 10(4):318-24. DOI:10.3109/19390211.2013.830673
[Show abstract][Hide abstract] ABSTRACT: arNOX, an age-related NADH oxidase, has been shown to play a role in the formation of the autophagosome. The role of autophagy in tumorigenesis and tumor proliferation led to the hypothesis that abnormal arNOX levels might play a role in the pathology of cancer. Serum samples from cancer patients and age- and gender-matched healthy controls were assayed to measure arNOX activity by a standard assay involving measurements of superoxide production based on the reduction of ferricyanide c by superoxide monitored from the increase in absorbance at 550 nm with reference at 540 nm. Superoxide dismutase was added to the end of the assay to ensure return of the rate of ferricytochrome c reduction to the base line. Cancer and cardiovascular diseases are inversely correlated. Similarly, arNOX levels were decreased in cancer patients when compared to non-cancer control subjects while high arNOX levels have been implicated as an important cause of LDL oxidation and increased risk of cardiovascular disease. These findings may help further the understanding of the pathologies of cancer and cardiovascular disease and have implications for risk assessment for both diseases as well as for future therapeutic intervention strategies.
Journal of medicine and life 07/2013; 1(2):38. DOI:10.14511/jlm.2013.010204
[Show abstract][Hide abstract] ABSTRACT: Age-related NADH oxidase (arNOX) is a cell surface protein shed into the circulation and other body fluids, which generates superoxide. The activity increases with age in human tissues and body fluids (serum, saliva, and perspiration) and is a potential source of age-related oxidative damage. We measured arNOX activity in the coelomic fluid of sea urchin species with different life spans. Coelomic fluid of long-lived sea urchin species Strongylocentrotus purpuratus and Strongylocentrotus franciscanus exhibited low levels of arNOX compared to the short-lived urchin species Lytechinus variegatus. arNOX activity was positively correlated with animal size in L. variegatus, whereas with S. purpuratus and S. franciscanus, arNOX activity and animal size were inversely correlated. The inverse correlation of arNOX activity with life span and decreased levels of arNOX with age in the long-lived species is consistent with a contribution of reduced arNOX activity to slower aging.
International Aquatic Research 01/2013; 5(1). DOI:10.1186/2008-6970-5-2
[Show abstract][Hide abstract] ABSTRACT: The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism.
The international journal of biochemistry & cell biology 06/2012; 44(11):1834-8. DOI:10.1016/j.biocel.2012.06.032 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The homodimeric, growth-related and time-keeping constitutive hydroquinone oxidase ENOX1 of the eukaryotic cell surface capable of oxidizing extracellular NAD(P)H and intracellular hydroquinones exhibits properties of the ultradian driver of the biological 24-h circadian clock by exhibiting a complex 2 + 3 set of oscillations with a period length of 24 min (repeats 60 times over 24 h). The oscillations require bound copper, are recapitulated by aqueous solutions of copper salts and appear to derive from 30 to 40 s periodic variations in the ratios of ortho and para nuclear spins of the paired hydrogen atoms of the elongated octahedral structure of the ENOX1 protein-bound copperII hexahydrates. By functioning in the manner of limit oscillators, these 30–40 s oscillations appear to generate the 24-min periodicity in the manner of a carrier wave. The ortho–para oscillations are highly synchronous through self-generated very low frequency electromagnetic fields which also serve to phase ENOX1/copperII oscillations. The synchronized populations of water molecules give rise to oscillatory electromagnetic fields that apparently are perceived by adjacent water molecules to create a collectively coherent synchronous system capable of extending over very long distances. The period length of the ENOX oscillations is temperature independent and entrained by light, melatonin, lithium, and caffeine. COS cells transformed with specific ENOX variants where specific cysteine codons are replaced by alanine codons yield circadian periods of 22, 34, or 42 h respectively, based on activity of glyceraldehyde-3-dehydrogenase in response to ENOX oscillations with period lengths of 22, 36, or 42 min. That the oscillations result from physical rather than chemical events accounts for the temperature independence of the period length of clock-related phenomena. Also, the period length of both the ENOX and copperII oscillations in D2O is increased to 30 min in keeping with the 30-h circadian day exhibited by organisms grown in deuterium oxide. An opportunity to link the ultradian ENOX oscillation to downstream activation of clock genes may be provided by responses of transcriptional factors to levels of NADH and especially to NAD+ generated by ENOX oscillations. Adverse effects resulting from chronic disturbances or disruptions of circadian rhythms by repeated rephrasing of the ENOX cycle may include an increased risk of cancer.
[Show abstract][Hide abstract] ABSTRACT: A homodimeric, growth-related and time-keeping hydroquinone oxidase
(ENOX1) of the eukaryotic cell surface capable of oxidizing extracellular NADH
exhibits properties of the ultradian driver of the biological 24 h circadian clock
by exhibiting a complex 2 + 3 set of oscillations with a period length of 24 min.
The oscillations require bound copper, are recapitulated by aqueous solutions of
copper salts and appear to derive from 30 to 40 sec periodic variations in the
ratios of ortho and para nuclear spins of the paired hydrogen atoms of the
elongated octahedral structure of the ENOX2 protein bound copperII
hexahydrates. Based on a heartbeat model of limit cycle oscillations, these
oscillations correlate with generation of the 24 min periodicity as a form of
carrier wave. A corollary of these observations is that the ortho-para oscillations
must occur in a highly synchronized manner. Attendant oscillatory changes in
redox potential offer an opportunity to monitor oscillations in synchronous
populations of water molecules. The periodicity of the ENOX1/copper/water
clock can be phased by brief 10 to 20 sec exposure to very low frequency
electromagnetic fields. The synchronized populations of oscillating water
molecules give rise to oscillatory electromagnetic fields that apparently are
perceived by adjacent water molecules to create a collectively coherent
synchronous system. Two asynchronous water samples placed adjacent to one
another but separated by a thin non-metal barrier become fully synchronized in a
matter of several min. A barrier of metal foil prevents the synchronization. The
corollary of these observations is that contiguous water molecules function
synchronously perhaps even over relatively long distances. Two samples of
water from contiguous still or flowing bodies of water collected from different
locations and analyzed simultaneously were synchronous in their oscillations in
redox potential measured as changes in rates of NADH oxidation. Our findings
suggest that water molecules communicate with each other via very low
frequency electromagnetic fields.
[Show abstract][Hide abstract] ABSTRACT: The key role of coenzyme Q (ubiquinone or Q) is in mitochondrial and prokaryotic energetics. Less well investigated is the basis for its presence in eukaryotic membrane locations other than mitochondria and in plasma where both antioxidant and potentially more targeted roles are indicated. Included in the latter is that of a lipid-soluble electron transfer intermediate that serves as the transmembrane component of plasma membrane and Golgi apparatus electron transport, which regulates cytosolic NAD(+) /NADH ratios and is involved in vectorial membrane displacements and in the regulation of cell growth. Important protective effects on circulating lipoproteins and in the prevention of coronary artery disease ensue not only from the antioxidant role of CoQ(10) but also from its ability to directly block protein oxidation and superoxide generation of the TM-9 family of membrane proteins known as age-related NADH oxidase or arNOX (ENOX3) and their shed forms that appear after age 30 and some of which associate specifically with low-density lipoprotein particles to catalyze protein oxidation and crosslinking.
[Show abstract][Hide abstract] ABSTRACT: HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.