Mutator/Hypermutable Fetal/Juvenile Metakaryotic Stem Cells and Human Colorectal Carcinogenesis

Adult age-specific colorectal cancer incidence rates increase exponentially from maturity, reach a maximum, then decline in extreme old age. Armitage and Doll (1) postulated that the exponential increase resulted from “n” mutations occurring throughout adult life in normal “cells at risk” that initiated the growth of a preneoplastic colony in which subsequent “m” mutations promoted one of the preneoplastic “cells at risk” to form a lethal neoplasia. We have reported cytologic evidence that these “cells at risk” are fetal/juvenile organogenic, then preneoplastic metakaryotic stem cells. Metakaryotic cells display stem-like behaviors of both symmetric and asymmetric nuclear divisions and peculiarities such as bell shaped nuclei and amitotic nuclear fission that distinguish them from embryonic, eukaryotic stem cells. Analyses of mutant colony sizes and numbers in adult lung epithelia supported the inferences that the metakaryotic organogenic stem cells are constitutively mutator/hypermutable and that their contributions to cancer initiation are limited to the fetal/juvenile period. We have amended the two-stage model of Armitage and Doll and incorporated these several inferences in a computer program CancerFit v.5.0. We compared the expectations of the amended model to adult (15–104 years) age-specific colon cancer rates for European-American males born 1890–99 and observed remarkable concordance. When estimates of normal colonic fetal/juvenile APC and OAT gene mutation rates (∼2–5 × 10[superscript −5] per stem cell doubling) and preneoplastic colonic gene loss rates (∼8 × 10[superscript −3]) were applied, the model was in accordance only for the values of n = 2 and m = 4 or 5.United Therapeutics Corporatio

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development

Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats

Normal pregnancy is associated with reduced blood pressure (BP) and decreased pressor response to vasoconstrictors, even though the renin-angiotensin system is upregulated. Angiotensin II (ANG II) activates both angiotensin type 1 receptors (AT1Rs) and angiotensin type 2 receptors (AT2Rs). Although the role of the AT1R in vascular contraction is well documented, the role of the AT2R in vascular relaxation, particularly during pregnancy, is less clear. It was hypothesized that the decreased BP and vasoconstriction during pregnancy was, at least in part, due to changes in AT2R amount, distribution, and/or postreceptor mechanisms of vascular relaxation. To test this hypothesis, systolic BP was measured in virgin and pregnant (day 19) Sprague-Dawley rats. Isometric contraction/relaxation was measured in isolated aortic rings, and nitric oxide (NO) production was measured using 4-amino-5-methylamino-2′,7′-difluorescein fluorescence. AT1R and AT2R mRNA expression and protein amount were measured in tissue homogenates using real-time RT-PCR and Western blots, and their local distribution was visualized in cryosections using immunohistochemistry and immunofluorescence. BP was lower in pregnant than virgin rats. Phenylephrine (Phe) caused concentration-dependent contraction that was reduced in the aorta of pregnant compared with virgin rats. Treatment with the AT2R antagonist PD-123319 caused greater enhancement of Phe contraction, and the AT2R agonist CGP-42112A caused greater relaxation of Phe contraction in the aorta of pregnant than virgin rats. ANG II plus the AT1R blocker losartan induced greater NO production in the aorta of pregnant than virgin rats. RT-PCR revealed increased mRNA expression of vascular endothelial NO synthase (eNOS), little change in AT1Rs, and increased AT2Rs in pregnant compared with virgin rats. Western blots revealed an increased protein amount of activated phospho-eNOS, little change in AT1Rs, and increased AT2Rs in pregnant compared with virgin rats. Immunohistochemistry and immunofluorescence analysis in aortic sections of virgin rats revealed abundant AT1R staining in tunica media that largely colocalized with actin in vascular smooth muscle and less AT2Rs mainly in the tunica intima and endothelium. In pregnant rats, AT1R staining in the smooth muscle layer and adventitia was reduced, and endothelial AT2R staining was enhanced. These data suggest an enhanced AT2R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT2Rs and increased postreceptor activated phospho-eNOS, which may contribute to the decreased BP during pregnancy

Palm Fruit Bioactives augment expression of Tyrosine Hydroxylase in the Nile Grass Rat basal ganglia and alter the colonic microbiome

Tyrosine hydroxylase (TH) catalyzes the hydroxylation of L-tyrosine to L-DOPA. This is the rate-limiting step in the biosynthesis of the catecholamines - dopamine (DA), norepinephrine (NE), and epinephrine (EP). Catecholamines (CA) play a key role as neurotransmitters and hormones. Aberrant levels of CA are associated with multiple medical conditions, including Parkinson's disease. Palm Fruit Bioactives (PFB) significantly increased the levels of tyrosine hydroxylase in the brain of the Nile Grass rat (NGR), a novel and potentially significant finding, unique to PFB among known botanical sources. Increases were most pronounced in the basal ganglia, including the caudate-putamen, striatum and substantia nigra. The NGR represents an animal model of diet-induced Type 2 Diabetes Mellitus (T2DM), exhibiting hyperglycemia, hyperinsulinemia, and insulin resistance associated with hyperphagia and accelerated postweaning weight gain induced by a high-carbohydrate diet (hiCHO). The PFB-induced increase of TH in the basal ganglia of the NGR was documented by immuno-histochemical staining (IHC). This increase in TH occurred equally in both diabetes-susceptible and diabetes-resistant NGR fed a hiCHO. PFB also stimulated growth of the colon microbiota evidenced by an increase in cecal weight and altered microbiome.  The metabolites of colon microbiota, e.g. short-chain fatty acids, may influence the brain and behavior significantly

Oil Palm Phenolics Inhibit the n Vitro Aggregation of β-Amyloid Peptide into Oligomeric Complexes

Alzheimer’s disease is a severe neurodegenerative disease characterized by the aggregation of amyloid-β peptide (Aβ) into toxic oligomers which activate microglia and astrocytes causing acute neuroinflammation. Multiple studies show that the soluble oligomers of Aβ42 are neurotoxic and proinflammatory, whereas the monomers and insoluble fibrils are relatively nontoxic. We show that Aβ42 aggregation is inhibited in vitro by oil palm phenolics (OPP), an aqueous extract from the oil palm tree (Elaeis guineensis). The data shows that OPP inhibits stacking of β-pleated sheets, which is essential for oligomerization. We demonstrate the inhibition of Aβ42 aggregation by (1) mass spectrometry; (2) Congo Red dye binding; (3) 2D-IR spectroscopy; (4) dynamic light scattering; (5) transmission electron microscopy; and (6) transgenic yeast rescue assay. In the yeast rescue assay, OPP significantly reduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptides. The data shows that OPP inhibits (1) the aggregation of Aβ into oligomers; (2) stacking of β-pleated sheets; and (3) fibrillar growth and coalescence. These inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammation and neuronal death and thereby may play some role in the prevention or treatment of Alzheimer’s disease

Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

Neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, are becoming more prevalent and an increasing burden on society. Neurodegenerative diseases often arise in the milieu of neuro-inflammation of the brain. Reactive astrocytes are key regulators in the development of neuro-inflammation. This study describes the effects of Palm Fruit Bioactives (PFB) on the behavior of human astrocytes which have been activated by IL-1β. When activated, the astrocytes proliferate, release numerous cytokines/chemokines including TNFα, RANTES (CCL5), IP-10 (CXCL10), generate reactive oxygen species (ROS), and express specific cell surface biomarkers such as the Intercellular Adhesion Molecule (ICAM), Vascular Cellular Adhesion Molecule (VCAM) and the Neuronal Cellular Adhesion Molecule (NCAM). Interleukin 1-beta (IL-1β) causes activation of human astrocytes with marked upregulation of pro-inflammatory genes. We show significant inhibition of these pro-inflammatory processes when IL-1β-activated astrocytes are exposed to PFB. PFB causes a dose-dependent and time-dependent reduction in specific cytokines: TNFα, RANTES, and IP-10. We also show that PFB significantly reduces ROS production by IL-1β-activated astrocytes. Furthermore, PFB also reduces the expression of ICAM and VCAM, both in activated and naïve human astrocytes in vitro. Since reactive astrocytes play an essential role in the neuroinflammatory state preceding neurodegenerative diseases, this study suggests that PFB may have a potential role in their prevention and/or treatment

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development