Estrogen is neuroprotective against hypoglycemic injury in murine N38 hypothalamic cells.

Estrogen (E2) has been demonstrated to possess protective effects from hypoglycemic toxicity, particularly in the pancreas. In the central nervous system, several brain regions, such as the hypothalamus, are highly vulnerable to hypoglycemic injuries that may lead to seizures, coma, and mortality. The present study performed a novel in vitro assay of hypoglycemic injury to hypothalamic cells, and is the first study, to the best of our knowledge, to demonstrate that E2 protects hypothalamic cells from hypoglycemic toxicity. The toxic effects of hypoglycemia on hypothalamic cells in vitro was determined by performing cell counts, together with MTT and lactate dehydrogenase assays, using the N38 murine hypothalamic cell line. Following 24 and 48 h in hypoglycemic conditions, a 60 and 75% reduction in cell number and mitochondrial function was observed, which reached 80 and ~100% by 72 and 96 h, respectively. E2 treatment prevented the hypoglycemia­induced loss in cell number and mitochondrial toxicity at 24 and 48 h. However at 72 and 96 h of hypoglycemic conditions, the neuroprotective effects of E2 on cell number or mitochondrial function was not significant or not present at all. In order to determine whether E2 exerted its effects through the AKT signaling pathway, the expression of proline­rich AKT substrate of 40 kDa (PRAS40) was analyzed. No alterations in PRAS40 expression were observed when N38 cells were exposed to hypoglycemic shock. From the biochemical and molecular data obtained, the authors speculated that E2 exhibits neuroprotective effects against hypoglycemic shock in hypothalamic cells, which dissipates with time. Despite demonstrating no significant effect on total AKT/PRS40 activity, it is possible that E2 may mediate these neuroprotective effects by upregulating the phosphorylated­AKT/pPRAS40 signaling pathway. The present study presented, to the best of our knowledge, the first in vitro model for hypoglycemic toxicity to hypothalamic cells, and provided evidence to suggest that E2 may protect hypothalamic cells from the damaging effects of hypoglycemia.

Burrowing in the freshwater mussel Elliptio complanata is sexually dimorphic and feminized by low levels of atrazine.

The widely used herbicide atrazine (ATR) may have endocrine-associated adverse effects, including on behavior. In this study, 120 adult freshwater mussels, Elliptio complanata, were exposed to ATR at the environmentally relevant concentrations of 1.5, 15, or 150 µg/L. Burrowing depth was evaluated hourly for 6 h and at sacrifice animals were sexed by gonad smear. Female controls burrowed overall approximately 30% less than males, the first report of sexual dimorphism in this behavior. Atrazine at 15 µg/L feminized burrowing in both sexes, in that exposed animals burrowed 20% less than their same-sex controls. Males treated with 1.5 µg /L ATR displayed approximately 20-fold higher vitellogenin (VTG) levels than same-sex controls. Higher concentrations of ATR were not associated with increasing effects. A scatterplot showed a weak binomial curve associating low burrowing with high VTG levels. Taken together, these data suggest a nonlinear dose response in behavioral and physiological feminization produced by ATR and support the need to reconsider the widespread use of this compound.

Dietary selenium and selenoprotein function.

Selenium is a trace mineral and an essential nutrient in the human diet. Selenium is found in soil and water and consequently enters the food chain through the root ways of plants and aquatic organisms. Some areas of the world are low in soil selenium resulting in a selenium deficient population and the appearance of an associated heart disease and bone disorders that can be corrected with dietary selenium. Indeed the requirement for dietary selenium was established by these observations and while selenium deficiency is rare in the West, patients requiring long-term intravenous feedings have also show heart disease associated with a deficiency of selenium in the feeding fluids. Subsequently, it has been established that dietary selenium can improve a wide range of human health conditions even in areas with soil replete in selenium.

Dietary and botanical anxiolytics.

Drugs used to treat anxiety have many negative side effects including addiction, depression, suicide, seizures, sexual dysfunction, headaches and more. Anxiolytic medications do not restore normal levels of neurotransmitters but instead manipulate the brain chemistry. For example, selective serotonin reuptake inhibitors (SSRIs) prevent the reuptake of serotonin from the synapse allowing serotonin to remain in the area of activity for a longer period of time but does not correct the lack of serotonin production. Benzodiazepines, such as Valium and Xanax®, stimulate GABA receptors, thus mimicking the calming effects of GABA but again do not fix the lack of GABA production. Often, the brain becomes accustomed to these medications and they often lose their effectiveness, requiring higher doses or different drugs. In contrast to anxiolytic drugs, there are herbs and nutrients which can stimulates neurotransmitter synthesis and more naturally effect and even adjust brain chemistry in the absence of many of the side effects experienced with drugs. Therefore this paper explores several herbal and nutritional approaches to the treatment of anxiety.

The antimicrobial and phagocytic defense mechanisms of the terrestrial gastropod Lehmannia nyctelia against Escherichia coli and Staphylococcus aureus.

BACKGROUND: Gastropods are significant vectors of human infectious diseases and are exposed to a wide range of soil parasites and micro-organisms. To defend against infection, we hypothesized that the slug produces both soluble and cellular defense mechanisms. MATERIAL/METHODS: In the gastropod Lehmannia nyctelia, soluble antimicrobial activity was measured by disk diffusion assay and phagocytosis was measured by in vitro and in vivo uptake of fluorescent Escherichia coli bioparticles. RESULTS: In disk diffusion assays we failed to observe either methanol or dimethylsulfoxide soluble material in the slug secretions and tissues. However, isolation of the slug skin cells demonstrated phagocytic morphology in vitro when exposed to Escherichia coli and Staphylococcus aureus and internalization of fluorescent Escherichia coli bioparticles. Further, examination of histological sections of a slug exposed to fluorescent Escherichia coli bioparticles revealed a subset of skin cells which had cytoplasmic accumulations of the bioparticles. CONCLUSIONS: These data suggest that the Lehmannia nyctelia skin contains active phagocytic cells that may affect the set of parasites and microbes for which slugs can serve as vectors of disease.

Formulations of dietary supplements and herbal extracts for relaxation and anxiolytic action: Relarian.

Dietary supplements are widely used for desired effects on memory, insomnia, mood and anxiety. This review focuses on supplements which have anxiolytic or mild relaxation properties and enhance mood. For example, Kava (Piper methysticum) is reported to have anaxiolytic actions and to reduce tension through skeletal muscle relaxation. Dried passion flower (genus Passiflora) is reported to reduce insomnia and hysteria. Skullcap (genus Scutellaria), hops (Humulus lupulus), lemon balm (Melissa officinalis) and Valerian (Valeriana officinalis) root are all herbs reported as anaxiolytic calming agents. Further, extracts of Magnolia and Phellondendron bark are mild sedatives. Supplements such as gamma-aminobutyric acid (GABA), theanine, tryptophan and 5-hydroxytryptophan (5-HTP) are reported to promote relaxation. In general, these supplements appear to act as GABA receptor agonists or to boost GABA levels, although Kava inhibits both norephinephrine uptake and sodium and potassium channels and 5-HTP may act through elevation of serotonin. While questions remain in the literature regarding the medicinal value of these supplements in treating mood and anxiety disorders, based on cellular and animal studies as well as human clinical trials the literature supports a role for these preparations as useful alternatives in the management of the stress and anxiety of everyday life.

The hemicellulose preparation, Natramune (PDS-2865), increases macrophage phagocytosis and nitric oxide production and increases circulating human lymphocytes levels.

BACKGROUND: Hemicellulose containing nutritional supplements demonstrate benefits to the immune system in vitro and in vivo. Here we show that Natramune (PDS-2865) stimulates phagocytosis, nitric oxide production and boosts viability in J774A.1 murine machrophages. We also show that dietary supplementation with Natramune (PDS-2865) significantly increases the levels of circulating lymphocytes in human subjects. MATERIAL/METHODS: In order to measure the beneficial effects of Natramune (PDS-2865) on cells of the immune system, phagocytosis was measured by J774A.1 uptake of fluorescently labeled E. Coli bioparticles, nitric oxide production was measured by the formation of nitrite, and cell proliferation and viability was measured by NADH reduction of WST-8. The effect of Natramune (PDS-2865) on human circulating leukocyte levels was measured in 18 volunteers after an 8 week regimen of two 250 mg doses daily after which blood was collected and blood cell number and types were counted. RESULTS: Natramune (PDS-2865) stimulated phagocytosis, nitric oxide production and promoted proliferation/viability in J774A.1 cells by 65%, 517%, and 155% respectively. Further, Natramune (PDS-2865) did boost human circulating total lymphocyte levels (18%) in a statistically significant manner and while all lymphocyte subtype levels also increased, the individual subtype increases were not statistically significant. CONCLUSIONS: Dietary supplementation with Natramune (PDS-2865) enhances immune system function and vitality.

Natramune and PureWay-C reduce xenobiotic-induced human T-cell alpha5beta1 integrin-mediated adhesion to fibronectin.

BACKGROUND: In vitro and in vivo studies demonstrate that nutritional supplementation reduces inflammation and inflammatory markers associated with T-cell adhesion mechanisms. Here, we investigate the effects of the nutritional supplements, Natramune (PDS-2865) and PureWay-C, on xenobiotic-induced alpha5beta1 integrin-mediated T-cell adhesion to fibronectin. MATERIAL/METHODS: The human CD4+ lymphoblastoid cell line CEM SS was treated with combinations of bifenthrin, blocking antibodies to human beta1 and alpha5 integrin, and nutrient supplements. After 30 minutes unattached cells were aspirated and the percent of attached cells was determined. RESULTS: Bifenthrin stimulated T-cell adhesion to fibronectin at concentrations between 1.0 and 100 microM with a maximal stimulation of 8.3-fold at 10 microM. At 500 microg/ml, Natramune reduced 100 microM bifenthrin-induced adhesion by nearly 90%. PureWay-C reduced by 1.5-fold the level of T-cell adhesion stimulated by bifenthrin concentrations of both 10 microM and 100 microM. The combination of Natramune and PureWay-C resulted in a 6.3 and 7.5-fold inhibition at 10 microM and 100 microM bifenthrin respectively. Antibody blocking studies demonstrated that bifenthrin induced CEM SS adhesion to fibronectin is mediated through alpha5beta1 integrin. Inhibition of T-cell adhesion achieved by anti-integrin antibodies was further reduced with 50 and 500 microg/ml Natramune treatment. Pretreatment of fibronectin with Natramune did not alter induced T-cell adhesion to fibronectin. CONCLUSIONS: These data demonstrate that xenobiotic-induced alpha5beta1 integrin mediated T-cell adhesion to fibronectin is reduced by nutritional supplementation with Natramune (PDS-2865) and PureWay-C. These data suggest the possibility that inflammatory responses associated with exposure to pollutants can be mitigated by nutritional supplementation.

Absorption rates and free radical scavenging values of vitamin C-lipid metabolites in human lymphoblastic cells.

BACKGROUND: In this study we investigated the cellular absorption rates, antioxidant and free radical scavenging activity of vitamin C-lipid metabolites. The absorption was measured in a human lymphoblastic cell line using a spectrophotometric technique. MATERIAL/METHODS: Cellular vitamin C levels in the human lymphoblastic H9 cell line were measured using the 2,4-dinitrophenylhydrazine spectrophotometric technique. Free radical scavenging activity of vitamin C-lipid metabolites was measured by the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) to 1,1-diphenyl-2-picryl hydrazine. Vitamin C-lipid metabolite scavenging of peroxyl radical oxygen reactive species (ORAC) was determined by fluorescence spectrophotometry. RESULTS: Compared to ascorbic acid (AA), calcium ascorbate (CaA), and calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C), vitamin C-lipid metabolites (PureWay-C) were more rapidly absorbed by the H9 human T-lymphocytes. The vitamin C-lipid metabolites (PureWay-C) also reduced pesticide-induced T-lymphocyte aggregation by 84%, while calcium ascorbate-calcium threonate-dehydroascorbate (Ester-C) reduced aggregation by only 34%. The vitamin C-lipid metabolites (PureWay-C) demonstrated free radical scavenging activity of nearly 100% reduction of DPPH at 20 microg/ml and oxygen radical scavenging of over 1200 micro Trolox equivalents per gram. CONCLUSIONS: These data demonstrate that the vitamin C-lipid metabolites (PureWay-C) are more rapidly taken-up and absorbed by cells than other forms of vitamin C, including Ester-C. This increased rate of absorption correlates with an increased protection of the T-lymphocytes from pesticide toxicities. Further, vitamin C-lipid metabolites (PureWay-C) are a potent antioxidant and have significant free radical scavenging capabilities.

A novel vitamin C preparation enhances neurite formation and fibroblast adhesion and reduces xenobiotic-induced T-cell hyperactivation.

BACKGROUND: Vitamin C (ascorbic acid, ascorbate) has been shown to enhance neurite outgrowth, promote fibroblast adhesion during wound healing, and reduce xenobiotic-induced leukocyte hyperactivity and inflammatory damage. In this study, a comparison was made between Ester-C and PureWay-C on these various cellular activities. MATERIAL/METHODS: PC12 cells were stimulated to form neurites with nerve growth factor, NIH 3T3 fibroblasts were seeded on fibronectin and H9 T-cells were stimulated to aggregate with the pyrethroid pesticide bifenthrin. The rate of neurite formation, fibroblast adhesion and T-cell homotypic aggregation was then measured in the absence and presence of various formulations of vitamin C including Ester-C and PureWay-CTM. RESULTS: With PureWay-C treatment, 12% of PC12 cells extended neurites within one hour of treatment and 45% of the cells extended neurites by hour nine. With Ester-C, 0% and 15% extended neurites at one and nine hours, respectively. NIH-3T3 fibroblast adhesion to fibronectin was enhanced by 4.7-fold with a 30 minute PureWay-CTM treatment while Ester-C increased fibroblast adhesion by only 1.5 fold. Further, PureWay-CTM reduced pesticide-mediated T-cell homotypic aggregation by 83% within 30 minutes of treatment while the reduction seen with Ester-C was only 33%. CONCLUSIONS: These data confirm the previous observations that vitamin C supplementation can promote neurite outgrowth, increase fibroblast adhesion and reduce xenobiotic induce immunocytes aggregation. More importantly, these data show that PureWay-C has a faster and greater beneficial effect on these parameters when compared to other vitamin C formulations.

Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways.

During neuromuscular junction formation, extracellular matrix-mediated signals cause muscle surface acetylcholine receptors (AChRs) to aggregate at synaptic sites. Two extracellular matrix proteins, agrin and laminin, have each been shown to initiate signaling pathways that culminate in AChR clustering in cultured muscle cells. Here we present evidence that laminin-induced AChR clustering is mediated by the activation of the Rho GTPases Cdc42, Rac and Rho. Clustering in response to laminin is blocked by the dominant negative mutants Cdc42N17, RacN17 and RhoN19, as well as by the Rho inhibitor C3 transferase. Moreover, laminin-induced AChR clustering is impaired by the Rho kinase inhibitor Y-27632. Agrin-induced AChR clustering has previously been shown to require activation of Cdc42, Rac and Rho. Therefore, although agrin and laminin use distinct transmembrane receptors to initiate AChR clustering, their signaling pathways converge at the level of Rho GTPase activation.

PolicosanolPlus and Neuroprevin ameliorate pesticide-mediated inhibition of neurite outgrowth and neurite degeneration.

BACKGROUND: Pesticide exposure is a recognized risk factor for neurodegenerative diseases. Recently, bifenthrin, a pyrethroid pesticide, was shown to inhibit the formation of neurites and cause neurite retraction, raising concern that these newer and less toxic pesticides may also contribute to neurodegenerative diseases. PolicosanolPlus and Neuroprevin are nutraceutical supplements which promote the survival of neurites in neuronal cell cultures. Here we determine if PolicosanolPlus and Neuroprevin can ameliorate the neurodegenerative effects of bifenthrin. MATERIAL/METHODS: PC12 cells were treated with NGF, bifenthrin, PolicosanolPlus and Neuroprevin in various combinations and the formation of neurites was assessed microscopically at times ranging from 12 to 72 hours post treatment. Bifenthrin was also withheld at the time of NGF, PolicosanolPlus and Neuroprevin treatment and added after neurite formed to assess neurite retraction. RESULTS: Bifenthrin (1 x 10(-6) M) inhibits neurite outgrowth, in the absence of cell death, by more than 50% at 12 hours and by more than 80% at 72 hours. With addition of PolicosanolPlus and/or Neuroprevin at the time of cell seeding, bifenthrin does not inhibit neurite outgrowth. Addition of bifenthrin to differentiated cells results in a retraction of 90% of neurites, while those with PolicosanolPlus and Neuroprevin show no significant retraction of neurites. CONCLUSIONS: The pesticide, bifenthrin, inhibits neurite formation and causes neurite retraction. PolicosanolPlus and Neuroprevin are nutraceutical supplements which ameliorate the effects of bifenthrin on neurite outgrowth and retraction. Dietary supplementation with PolicosanolPlus and Neuroprevin may protect against developmental and long-term neurodegenerative events that result from exposure to pesticides.

Bifenthrin causes neurite retraction in the absence of cell death: a model for pesticide associated neurodegeneration.

BACKGROUND: Bifenthrin is a synthetic pyrethroid insecticide derivative of naturally occurring pyrethrins from chrysanthemum flowers. Bifenthrin is considered relatively safe and therefore incorporated as the active ingredient in preparations sold over the counter for household use. Recent studies have raised concern that chronic exposure to pesticides in the home setting may increase the risk for neurodegenerative diseases. To address this concer, in the present study, bifenthrin is added to pre-differentiated PC12 and effect of bifenthrin on the retraction of existing neurites is observed a model for neurodegeneration. MATERIAL/METHODS: PC12 cells were differentiated with nerve growth factor for twenty-four hours and then treated with what was determined to be a sublethal dose of bifenthrin for up to an additional 48 hours. The percent of cells with neurites was assessed at various times before and after nerve growth factor treatment. Bifenthrin toxicity was determined using trypan blue exclusion. RESULTS: Bifenthrin was not toxic to PC12 cells at concentrations ranging from 1 x 10(-10) M to 1 x 10(-4) M. Twenty-four hours after nerve growth factor treatment, a maximum percent of cells had formed neurites and with a treatment of 1 x 10(-5) M bifenthrin, approximately 80% of these neurites retracted in within 12 additional hours and almost all neurites had retracted within 48 hours. Trypan exclusion showed that these cells were viable. CONCLUSIONS: These data show that bifenthrin can stimulate the retraction of neurites in the absence of frank toxicity.

Bifenthrin inhibits neurite outgrowth in differentiating PC12 cells.

BACKGROUND: Bifenthrin is a third generation member of the synthetic pyrethroid family of insecticides. As a new pesticide within a relatively new class of pesticides, bifenthrin is considered relatively safe. Here, we used the PC12 neuronal cell line to examine the effect of bifenthrin on the formation of neurites and the potential developmental neurotoxicity of this pesticide. MATERIAL/METHODS: PC12 cells were exposed to varying concentrations of technical grade bifenthrin or Ortho Home Defense. Cell viability was determined using the AlmarBlue Toxicity Assay. Nontoxic concentrations of these chemicals were concomitantly with nerve growth factor and neurite outgrowth was assessed. RESULTS: Ortho Home Defense preparation reduced PC12 cell viability by approximately 50% and 70% at dilutions that correlate to bifenthrin concentrations of 10(-5) M and 10(-4) M, respectively. In contrast, technical grade bifenthrin, was not toxic to PC12 cells at 10(-3) M, which was the highest concentration tested that was soluble. At "nontoxic" concentrations of 10(-7) M and 10(-6) M, the Ortho Home Defense inhibited nerve growth factor-mediated neurite outgrowth by 30% and 55% respectively. Furthermore the nontoxic concentrations of technical grade bifenthrin of 10(-6) M and 10(-3) M inhibited neurite outgrowth by approximately 35% and 75% respectively. CONCLUSIONS: These data suggest that the toxicity of the Ortho Home Defense preparation was due to the "inert" additives in the preparation and not the bifenthrin itself. Further, these data suggest that, even in the absence of overt toxicity, bifenthrin may have deleterious effects to developing nervous system.

Bifenthrin activates homotypic aggregation in human T-cell lines.

BACKGROUND: Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. MATERIAL/METHODS: The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. RESULTS: Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. CONCLUSIONS: The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

Identification of cell binding sites in the laminin alpha5-chain G domain.

The laminins consist of at least 11 polypeptides (5 alpha-chains, 3 beta-chains, and 3 gamma-chains) specific to basement membranes. Here we investigate the biological activity associated with the G domain of the newly identified laminin alpha5-chain using 113 overlapping synthetic peptides (positions 2679-3635). Using HT-1080 cells, 21 peptides showed attachment activity either on peptide-coated tissue culture plates or to peptide-conjugated Sepharose beads. Heparin inhibited cell attachment to 16 peptides, while ethylenediaminetetraacetic acid exhibited no inhibitory activity. Peptides A5G-27, A5G-65, and A5G-71 showed the strongest cell attachment, with the minimum active core sequences of the peptides being GIIFFL, HQNMGSVNVSV, and YLQFVG, respectively. Furthermore, these 16 peptides were tested for their ability to stimulate neurite outgrowth in the PC12 cells. A5G-3, A5G-33, A5G-71, A5G-73, A5G-81, and A5G-101 were the only peptides of the 16 that demonstrated the ability to promote neurite outgrowth. These results demonstrate that synthetic peptides with alpha5-chain G domain primary amino acid sequences possess some of the same biological activities attributable to the whole laminin and the alpha5-chain G domain. Therefore, these peptides may be useful in the investigation of laminin-receptor interactions and possibly mechanisms of laminin signal transduction.