It has been suggested that the CB1 G-protein-coupled receptor is internalized following agonist binding and activation of the second messenger pathways. The process of desensitization and resensitization is intimately involved with receptor internalization. Phosphorylation alters tolerance to cannabinoids thus contributing to tolerance. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. These findings led us to look at which kinase(s) may be involved in cannabinoid tolerance. We therefore hypothesize that by preventing phosphorylation of the CB1 receptor, we may reverse tolerance. We evaluated our hypothesis by testing the role of several kinases in tolerance: protein kinase A (PKA), protein kinase C (PKC), protein kinase C (PKG). Beta Adrenergic Receptor Kinase (β-ARK), Phosphatidylinositol 3-kinase (PI3K) and the src family tyrosine kinase. We also looked at cAMP and cGMP analogs. We evaluated PKA using KT5720, a PKA inhibitor; PKC using bisindolylmaleimide I, HCI] (bis), a PKC inhibitor; PKG using KT5823, a PKG inhibitor; β-ARK using Low molecular weight heparin (LMWH), a β-ARK inhibitor; PI3K using LY294002, a PI3K inhibitor and PP1 a src family tyrosine kinase inhibitor. The cAMP analog was dibutyryl-cAMP and the cGMP analog was dibutyryl-cGMP. ICR mice were rendered tolerant to △9- tetrahydrocannabinol (△9-THC) by administering injections of 20mg/kg △9-THC s.c. every 12 hours for 6.5 days. The mice were subsequently challenged 24 hours later with an ED8O of △9-THC at 20μg/mouse (i.t.). Antinociception was measured by the tail-flick test, %MPE’s and ED5O’s were calculated. The PKG inhibitor, KT5823, showed no significant change in %MPE. The β-ARK inhibitor, LMWH, showed no significant change in the %MPE. The PI3K inhibitor, LY294002, showed no significant change in the %MPE. Inhibition of PKC, by bis had no effect on tolerance, but at a higher dose attenuated the antinociceptive effect of △9-THC in non-tolerant mice. PPl, the src family tyrosine kinase inhibitor, reversed tolerance. KT5720, the PKA inhibitor reversed △9- THC tolerance. These data support a role for PKA and tyrosine kinase in phosphorylation events in THC tolerant mice. (Supported by NIDA grants K02DA00186 and P5ODA05274).

The activity of protein kinase C (PK-C) has been implicated in regulating the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C activators, phorbol 12,13- dibutyrate, mezerein, and bryostatin 1, on the proliferation and lineage commitment of enriched CD34+ human myeloid progenitor cells stimulated by lL-3, GM-CSF, stem cell factor and the lL-3/GM-CSF hybrid cytokine plXY321. Coadministration of these PK-C activators with plateau concentrations of rlL-3 or rGM-CSF induced 100-150% increase in the number of day 14 CFU-GM.with a selective stimulation on neutrophil and macrophage lineages while inhibiting eosinophilic growth. plXY321 stimulated an equivalent number of CFU-GM, including a predominant eosinophilic component, when compared to the combination of saturating levels of GM-CSF and lL-3. Bryostatin 1, when coadministered with plXY321 (or with the combination of lL-3 and GM-CSF), selectively enhanced the growth of neutrophilic and monocytic lineages while inhibiting eosinophil development. The inhibition of eosinophil colonies by bryostatin 1 was not mimicked by the coadministration of rSCF, rG-CSF or rCSF-1 with plXY321. Furthermore, neutralizing antibodies to rG-CSF and rCSF-1 failed to block potentiation of neutrophil or macrophage colony formation stimulated by bryostatin 1 in conjunction with plXY321, suggesting that accessory cell effects are not solely responsible for this phenomenona. rSCF synergistically enhanced plXY321 induced colony formation by an average of 144% by selectively stimulating neutrophilic and eosinophilic growth. Coadministration of bryostatin 1 with rSCF and plXY321 further increased colony formation by an average of 81%. This combination selectively stimulated cells of the macrophage lineage, and inhibited eosinophil differentiation. However, bryostatin 1 inhibited erythroid (BFU-E) and erythroid/myeloid mixed (CFU-GEMM) colonies induced by plXY321 alone or in combination with rSCF. Together these results indicate that 1) PK-C activity is involved in the growth and lineage commitment of early and committed myeloid progenitor cells. 2) Pharmacologic manipulation of PK-C may regulate the growth and differentiation of those cells exposed to early hematopoietic growth factors. This study raises the possibility that pharmacologic intervention at PK-C,in conjunction with hematopoietic growth factors, might be useful in the ex vivo expansion of hematopoietic progenitor cells.

Previous reports have indicated a dichotomy in the actions of TCDD on humoral immunity, both in vitro and in vitro, in which enhancements and suppression have been identified. The latter effect has been correlated with induction of liver P4501A1 enzyme activity, a response which is regulated by the Ah-gene locus. Additionally, the primary alteration in suppression of antibody responses is in the differentiation of the B cell. Therefore, the current investigation was undertaken to determine the relationship between these dual actions of TCDD on humoral immunity as related to its direct actions on B lymphocyte function. Specific emphasis was placed on determining the potential role of the Ah-gene locus in the modulation of the B cell and in vitro antibody responses. Initial observations determined that: 1) the degree of suppression of in vitro antibody responses in B6C3F1 mouse (an Ah-high responder strain) splenocytes is dependent on both the lot, concentration, and type (i.e., fetal bovine versus newborn bovine) of serum used in culture; and 2) there is a similarity in the actions of TCDD and Staphylococcus aureus Cowan strain I (SAC); a polyclonal B cell activator. These observations prompted the study of the direct effects of TCDD on B cells in different stages of in vivo activation and in the presence of different serum environments for which SAC has been reported to have opposing actions. TCDD was found to increase background levels of proliferation and differentiation in resting B cells (Go); thereby suggesting that resting B cells are a primary target in enhancement of immune responses by TCDD. However, no effect was seen on either response when the cells were stimulated with lipopolysaccharide (LPS). In cycling B cells (G1), TCDD caused suppression of both background and LPS-stimulated proliferation and differentiation and demonstrated a serum dependency that paralleled its actions in whole lymphocyte antibody responses; thereby demonstrating that it is the cycling B cell that is the primary target in suppression of antibody responses by TCDD and that both proliferation and differentiation are affected. The modulatory role of serum was also determined in primary hepatocyte cultures using P4501Al enzyme induction as the endpoint. Sera which supported either no effect or enhancements in both whole lymphocyte and purified B cell responses did not support induction of P4501A1 activity above BSA, a protein control. Conversely, sera which supported a consistent and dose-related suppression of Mug antibody responses were found to enhance the induction of P4501A1 activity. More importantly, normal mouse serum was found to allow for the full expression of the Ah-dependent phenomena in vitro, where primary hepatocytes and splenocytes from Ah-high responder (B6C3F1) and Ah-low responder (DBA/2) mouse strains were affected by TCDD in a manner that parallels the effects seen in the two strains following in vivo exposures. The latter results using mouse sera are consistent with a role by the Ah-gene locus in the direct effects of TCDD on whole lymphocyte and purified B cell antibody responses. However, the results with other, more traditional sources of sera, indicate that the ultimate expression of the TCDD- induced responses can be modulated by serum factors, which are at present unidentified. Furthermore, the results of this investigation indicate that the susceptibility of the cell, as related to its stage in the cell cycle, can contribute to the complex effects that are seen in the alteration of antibody responses following TCDD exposure.

Swamps are unique ecological communities that provide many valuable ecosystem services. In Kentucky, however, many swamps were altered by cypress removal and land development in their watersheds. Cypress Creek Swamp, which lies near Paducah in western Kentucky, is a good example of a swamp whose ecological integrity may be threatened by past and current nearby land use practices. This study was conducted to assess the water quality and macro- and microinvertebrate communities in the swamp. Three sites were monitored for temperature, dissolved oxygen, pH, specific conductivity, depth, phosphorus measured as orthophosphate, nitrite (NO2") and nitrate (NO3", NOx collectively), and ammonia nitrogen (NH3). The temperature, dissolved oxygen, NH3 and NOx concentrations changed with the growing season, but pH demonstrated little variability among the sites. The specific conductivity and phosphorus levels were highly variable. Principal component analysis (PCA) indicated no significant difference in microinvertebrate taxa identified among locations or through time. A oneway analysis of variance (ANOVA) indicated no significant difference in macroinvertebrate population total densities between locations (P = 0.847), and a oneway analysis of variance (ANOVA) showed no significant difference in microinvertebrate population total densities among locations (P = 0.153) or through time (P = 0.294). As development continues in the watershed, this work provides an important baseline for future water quality monitoring in the preserve.

The effectiveness of using constructed wetlands to remove unwanted nutrients, increase dissolved oxygen while at the same time decreasing the biological oxygen demand, and to reduce the levels of the Fecal Coliform Bacteria from a swine operation was evaluated. The indicator of proper waste purification will be the result of testing for the following: ammonia nitrogen, nitrate nitrogen, total phosphorus, total suspended solids, dissolved solids, dissolved oxygen, biological oxygen demand, and Fecal Coliform Bacteria. The wetland was divided into nine connected cells that covered approximately 3.8 hectares. Material was loaded from an anaerobic holding lagoon on four separate occasions during the testing period. As the material passed through the wetland, the vegetation, water column, substrate, and microbial populations functioned as the purification factors in the wetlands. During the sampling period, water was collected from each cell and analyzed for results. The data indicated that the constructed wetlands were effective in the waste management at a large swine production unit. Ammonia nitrogen showed an acceptable decrease, allowing nitrogen to be freed or converted into nitrate nitrogen. Total phosphorus and dissolved solids showed an expected decrease. Total suspended solids showed an overall decrease from the upper cells to the lower cells; however, results fluctuated during the testing period. Dissolved oxygen and biological oxygen demand showed an almost perfect inverse relationship with dissolved oxygen increasing as biological oxygen demand decreased. The removal of Fecal Coliform Bacteria was the most impressive, with the majority of bacteria being removed in the upper cells.

<p> The past several decades have witnessed a dramatic expansion in the rates of metabolic disease, most prominently the obesity and diabetes epidemics. While metabolic disease is undoubtedly driven by increased caloric intake and decreased physical activity, exposure to endocrine disrupting chemicals (EDCs) has been implicated as a causal factor in the development of metabolic disease. EDCs are exogenous compounds capable of modulating endogenous hormonal axes, with some compounds capable of interfering with metabolic pathways. Prior work identified the fungicide and booster biocide tolylfluanid (TF) as a potent EDC with the capacity to induce adipocyte differentiation and impair adipocyte insulin signaling through stimulation of the glucocorticoid receptor (GR). The present studies seek to expand upon these data by investigating the outcomes of dietary exposure to TF across the lifespan, with the hypothesis that TF disrupts energy metabolism through aberrant stimulation of the GR, and that disruptions in global metabolic homeostasis are driven by modulation of adipose physiology. When male mice were provided a diet supplemented with 100ppm TF, they exhibited several metabolic changes that mirror the metabolic syndrome, including augmented visceral adiposity, glucose intolerance, global and cellular insulin resistance, and disruptions in circadian rhythms. Importantly, gene set enrichment analysis identified an enrichment of GR-dependent genes in the adipose tissue of exposed mice. Next, investigating the interaction of TF with diet identified novel differences in the outcomes of exposure depending on the background macronutrient content of the diet. Finally, developmental exposure to TF during prenatal and early postnatal life was found to modulate insulin-glucose homeostasis in adult life, in a sex-dependent manner. Taken together, these findings identify TF as a novel metabolic disruptor <i> in vivo,</i> and support prior studies identifying TF as a potent environmental glucocorticoid.</p>

In the field of environmental epidemiology, there is a great deal of care required in constructing models that accurately estimate the effects of environmental exposures on human health. This is because the nature of the data that is available to researchers to estimate these effects is almost always observational in nature, making it difficult to adequately control for all potential confounders - both measured and unmeasured. Here, we tackle three different problems in which the goal is to accurately estimate the effect of an environmental exposure on various health outcomes.

<p> A Systemic Family Constellation is a phenomenological systemic group process that promotes healing and transformation in individuals through the use of representatives who stand in for family members, so that entanglements with the ancestors can be revealed and brought to reconciliation. Family Constellations were created by German psychotherapist and former priest, Bert Hellinger, who spent 16 years as a missionary and educator with the Zulu peoples of South Africa. Hellinger suggests that individuals become entangled with the fate of the ancestors. This study proposes that epigenetics can explain the heritability of ancestral experiences. </p><p> This study explores the role of epigenetics in the transgenerational transmission of the effects of trauma, experiences, and behaviors of the ancestors as observed in Systemic Family Constellations. It employs a multiparadigmatic model of research and performs a systematic review of existing literature on epigenetics from the fields of biology, genetics, medicine and psychology, and demonstrates that some epigenetic changes can be inherited for as many as four generations&mdash;and possibly iv more, without any changes in the underlying DNA. A systematic review of existing literature has become a viable research method in the fields of medicine and the social sciences in recent years, and is used here to explore epigenetic changes in genomic expression that are transmitted transgenerationally. This study recommends that epigenetics be added as a scientific explanation to the existing metaphysical theories of how constellations work, that include: (a) the knowing field, (b) morphic fields and morphic resonance, and (c) indigenous ways of knowing. </p><p> Some of the examples of epigenetic inheritance presented in this study, include the epigenetic effects of major traumas, such as the bombing of the World Trade Center, the Holocaust, and the Dutch Hunger Winter, as well as the effects of mothering on the stress responses of their offspring, the effects of feast or famine in utero that can potentially result in schizophrenia, and the effects of child sex abuse on mental health in adulthood and on same sex orientation&mdash;as well as potential evolutionary changes.</p>

A study on community exposure to Foray 48B, a biological insecticide, due to aerial spraying.

This report presents the results of the first phase of a study examining the occupational and environmental exposures of residents of a coastal British Columbia community to organochlorine compounds.