Summary
Research in biogerontology has largely focused on the complex relationship between mitochondrial dysfunction and biological aging. In particular, the mitochondrial free radical theory of ...aging (MFRTA) has been well accepted. However, this theory has been challenged by recent studies showing minimal increases in reactive oxygen species (ROS) as not entirely deleterious in nature, and even beneficial under the appropriate cellular circumstances. To assess these significant and nonintuitive observations in the context of a functional system, we have taken an in silico approach to expand the focus of the MFRTA by including other key mitochondrial stress response pathways, as they have been observed in the nematode Caenorhabditis elegans. These include the mitochondrial unfolded protein response (UPRmt), mitochondrial biogenesis and autophagy dynamics, the relevant DAF‐16 and SKN‐1 axes, and NAD+‐dependent deacetylase activities. To integrate these pathways, we have developed a multilevel hybrid‐modeling paradigm, containing agent‐based elements among stochastic system‐dynamics environments of logically derived ordinary differential equations, to simulate aging mitochondrial phenotypes within a population of energetically demanding cells. The simulation experiments resulted in accurate predictions of physiological parameters over time that accompany normal aging, such as the declines in both NAD+ and ATP and an increase in ROS. Additionally, the in silico system was virtually perturbed using a variety of pharmacological (e.g., rapamycin, pterostilbene, paraquat) and genetic (e.g., skn‐1, daf‐16, sod‐2) schemes to quantitate the temporal alterations of specific mechanistic targets, supporting insights into molecular determinants of aging as well as cytoprotective agents that may improve neurological or muscular healthspan.
Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of ...genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Melioidosis is caused by the facultative intracellular bacterium Burkholderia pseudomallei and is potentially fatal. Despite a growing global burden and high fatality rate, little is known about the ...disease. Recent studies demonstrate that cyclooxygenase-2 (COX-2) inhibition is an effective post-exposure therapeutic for pulmonary melioidosis, which works by inhibiting the production of prostaglandin E2 (PGE2). This treatment, while effective, was conducted using an experimental COX-2 inhibitor that is not approved for human or animal use. Therefore, an alternative COX-2 inhibitor needs to be identified for further studies. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) COX-2 inhibitor marketed outside of the United States for the treatment of migraines. While this drug was developed for COX-2 inhibition, it has been found to modulate other aspects of inflammation as well. In this study, we used RAW 264.7 cells infected with B pseudomallei to analyze the effect of TA on cell survival, PGE2 production and regulation of COX-2 and nuclear factor- kappaB (NF-ĸB) protein expression. To evaluate the effectiveness of post-exposure treatment with TA, results were compared to Ceftazidime (CZ) treatments alone and the co-treatment of TA with a sub-therapeutic treatment of CZ determined in a study of BALB/c mice. Results revealed an increase in cell viability in vitro with TA and were able to reduce both COX-2 expression and PGE2 production while also decreasing NF-ĸB activation during infection. Co-treatment of orally administered TA and a sub-therapeutic treatment of CZ significantly increased survival outcome and cleared the bacterial load within organ tissue. Additionally, we demonstrated that post-exposure TA treatment with sub-therapeutic CZ is effective to treat melioidosis in BALB/c mice.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide transcriptional response and causes a variety of adaptive and maladaptive physiological functions. Such functions are ...entirely dependent on the type of ligand activating it, and therefore, the nuances in the activation of this receptor at the single-cell level have become a research interest for different pharmacological and toxicological applications. Here, we investigate the activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine hepatoma cell line. The exogenous compounds analyzed produced different levels of ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and analyzed by both flow cytometric and computational simulation techniques. Interestingly, simulation experiments reported herein were able to reproduce and quantitate the natural single-cell stochasticity inherent to mammalian cell lines as well as the ligand-specific differences in ultrasensitivity. Classical AhR modulators 2,3,7,8-tetrachlorodibenzodioxin (10
− 1
–10
5
pM), PCB-126 (10
− 1
–10
7
pM), and benzoapyrene (10
− 1
–10
7
pM) produced the greatest levels of single-cell ultrasensitivity and most maximal responses, while consumption-based ligands indole-3-carbinol (10
3
–10
9
pM), 3,3′-diindolylmethane (10
3
–10
8
pM), and cannabidiol (10
3
–10
8
pM) caused low-level AhR activation in more purely graded single-cell fashions. All compounds were tested and analyzed over a 24 h period for consistency. The comparative quantitative results for each compound are presented within. This study aids in defining the disparity between different types of AhR modulators that produce distinctly different physiological outcomes. In addition, the simulation tool developed for this study can be used in future studies to predict the quantitative effects of diverse types of AhR ligands in the context of pharmacological therapies or toxicological concerns.
Alzheimer’s disease (AD) is the leading cause of dementia and one of the most common causes of death worldwide. As an age-dependent multifactorial disease, the causative triggers of AD are rooted in ...spontaneous declines in cellular function and metabolic capacity with increases in protein stressors such as the tau protein. This multitude of age-related processes that cause neurons to change from healthy states to ones vulnerable to the damage seen in AD are difficult to simultaneously investigate and even more difficult to quantify. Here we aimed to diminish these gaps in our understanding of neuronal vulnerability in AD development by using simulation methods to theoretically quantify an array of cellular stress responses and signaling molecules. This temporally-descriptive molecular signature was produced using a novel multimethod simulation approach pioneered by our laboratory for biological research; this methodology combines hierarchical agent-based processes and continuous equation-based modeling in the same interface, all while maintaining intrinsic distributions that emulate natural biological stochasticity. The molecular signature was validated for a normal organismal aging trajectory using experimental longitudinal data from
Caenorhabditis elegans
and rodent studies. In addition, we have further predicted this aging molecular signature for cells impacted by the pathogenic tau protein, giving rise to distinct stress response conditions needed for cytoprotective aging. Interestingly, our simulation experiments showed that oxidative stress signaling (via
daf-16
and
skn-1
activities) does not substantially protect cells from all the early stressors of aging, but that it is essential in preventing a late-life degenerative cellular phenotype. Together, our simulation experiments aid in elucidating neurodegenerative triggers in the onset of AD for different genetic conditions. The long-term goal of this work is to provide more detailed diagnostic and prognostic tools for AD development and progression, and to provide more comprehensive preventative measures for this disease.
High doses of the commonly used herbicide atrazine have been shown to suppress luteinizing hormone (LH) release. To determine whether atrazine alters the function of gonadotropin-releasing hormone ...(GnRH) neurons, we examined the effects of atrazine on GnRH neuronal activation and the subsequent release of LH normally associated with ovulation. Ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening using an indwelling right atrial cannula, and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. Results of these studies show that 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. To determine if the effects of atrazine were long lasting, additional studies were performed examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal. Within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal. These data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment.
Atrazine 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-tri-azine is one of the most commonly used herbicides in the United States. Atrazine has been shown to suppress luteinizing hormone (LH) release ...and can lead to a prolongation of the estrous cycle in the rat. The objectives of this study were to examine the effects of atrazine on normal tonic release of LH and to elucidate the site of action of atrazine in the hypothalamic-pituitary-gonadal axis. Episodic release of gonadotropin-releasing hormone (GnRH) and the corresponding release of LH from the anterior pituitary gland are required for normal reproductive function. To determine if atrazine affects pulsatile LH release, ovariectomized adult female Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle control for 4 days. On the final day of atrazine treatment, blood samples were obtained using an indwelling right atrial cannula. In the group receiving 200 mg/kg, there was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude. To determine if the effects of atrazine on LH release were due to changes at the level of the pituitary, animals were passively immunized against endogenous GnRH, treated with atrazine, and challenged with a GnRH receptor agonist. Atrazine failed to alter pituitary sensitivity to the GnRH receptor agonist at any dose used. Taken together, these findings demonstrate that high doses of atrazine affect the GnRH pulse generator in the brain and not at the level of gonadotrophs in the pituitary.
The mechanisms underlying cognitive and neurobehavioral abnormalities associated with childhood exposure to manganese (Mn) are not well understood but may be influenced by neuroinflammatory ...activation of microglia and astrocytes that results in nitrosative stress due to expression of inducible nitric oxide synthase (iNOS/NOS2). We therefore postulated that gene deletion of NOS2 would protect against the neurotoxic effects of Mn in vivo and in vitro. Juvenile NOS2 knockout (NOS2(-/-)) mice were orally exposed to 50 mg/kg of MnCl₂ by intragastric gavage from days 21 to 34 postnatal. Results indicate that NOS2(-/-) mice exposed to Mn were protected against neurobehavioral alterations, despite histopathological activation of astrocytes and microglia in Mn-treated mice in both genotypes. NOS2(-/-) mice had decreased Mn-induced formation of 3-nitrotyrosine protein adducts within neurons in the basal ganglia that correlated with protection against Mn-induced neurobehavioral defects. Primary striatal astrocytes from wildtype mice caused apoptosis in cocultured striatal neurons following treatment with MnCl₂ and tumor necrosis factor-α, whereas NOS2(-/-) astrocytes failed to cause any increase in markers of apoptosis in striatal neurons. Additionally, scavenging nitric oxide (NO) with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) prevented the ability of Mn- and cytokine-treated wildtype astrocytes to cause apoptosis in cocultured striatal neurons. These data demonstrate that NO plays a crucial role in Mn-induced neurological dysfunction in juvenile mice and that NOS2 expression in activated glia is an important mediator of neuroinflammatory injury during Mn exposure.
Canine and human osteosarcoma (OSA) have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time ...of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.
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