Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

Disparate effects of non-steroidal anti-inflammatory drugs on apoptosis in guinea-pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo-oxygenase-2 selective inhibitor, NS-398, on a primary culture of guinea-pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase-3-like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N-Hexanoyl-D-sphingosine (C6-ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase-3-like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6-ceramide. The inhibitory effect of diclofenac on basal caspase-3-like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase-3-like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase-3-like activity in cell homogenates and also inhibited human recombinant caspase-3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea-pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

Assessment of the astrogliotic responses of three human astrocytoma cell lines to ethanol, trimethyltin chloride and acrylamide

The astrogliotic responses of the CCF-STTG1, U251-MG, and U373-MG human astrocytoma lines were determined after exposure to ethanol, trimethyltin chloride (TMTC), and acrylamide over 4, 16, and 24 h. Basal glial fibrillary acidic protein (GFAP) expression in the U-251MG and U373-MG cells was 10-fold greater than the CCF-STGG1 line. Ethanol treatment over 24 h, but not at 4 and 16 h, resulted in significant increases in GFAP in all three glioma lines at sub-cytotoxic levels; the GFAP responses in the CCF-STTG1 line were the most sensitive, as concentrations of 0.1 and 1 mM led to increases in GFAP expression compared with control of 56.8 ± 15.7 and 58.9 ± 11.5%, respectively (P < 0.05). Treatment with TMTC (1 μM) over 4 h showed elevated GFAP expression in the U251-MG cell line to 28.0 ± 15.7% above control levels (P < 0.01), but not in the other U373-MG or CCF-STTG1 cells. At 4 h, MTT turnover was markedly increased compared with control, particularly in the U373-MG line at concentrations as low as 1 μM (17.1 ± 2.3%; P < 0.01). TMTC exposure over 16 and 24 h resulted in reduction in GFAP expression in all three lines at concentrations; at 24 h incubation, the reduction was >50% (P < 0.01). There were no changes in GFAP expression or MTT turnover in response to acrylamide except at the highest concentration ranges of 10-100 mM. This study underlines the significance of period of exposure, as well as toxin concentration in astrocytoma cellular response to toxic pressure. © 2007 Elsevier Ireland Ltd. All rights reserved.

The role of amyloid precursor protein in neuronal and non-neuronal cell lines

Models of Alzheimer’s disease (AD) have provided useful insights into the pathogenesis and mechanistic pathways that lead to its development. One emerging idea about AD is that it may be described as a hypometabolic disorder due to the reduction of glucose uptake in AD brains. Inappropriate processing of Amyloid Precursor Protein (APP) is considered central to the initiation and progression of the disease. Although the exact role of APP misprocessing is unclear, it may play a role in neuronal metabolism before the onset of neurodegeneration. To investigate the potential role of APP in neuronal metabolism, the SHSY5Y neuroblastoma cell line was used to generate cell lines that stably overexpress wild type APP695 or express Swedish mutated-APP observed in familial AD (FAD), both under the control of the neuronal promoter, Synapsin I. The effects of APP on glucose uptake, cellular stress and energy homeostasis were studied extensively. It was found that APP-overexpressing cells exhibited decreased glucose uptake with changes in basal oxygen consumption in comparison to control cell lines. Similar studies were also performed in fibroblasts taken from FAD patients compared with control fibroblasts. Previous studies found FAD-derived fibroblasts displayed altered metabolic profiles, calcium homeostasis and oxidative stress when compared to controls. As such, in this study fibroblasts were studied in terms of their ability to metabolise glucose and their mitochondrial function. Results show that FAD-derived fibroblasts demonstrate no differences in mitochondrial function, or response to oxidative stress compared to control fibroblasts. However, control fibroblasts treated with Aβ1-42 demonstrated changes in glucose uptake. This study highlights the importance of APP expression within non-neuronal cell lines, suggesting that whilst AD is considered a brain-associated disorder, peripheral effects in non-neuronal cell types should also be considered when studying the effects of Aβ on metabolism.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers

The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 µg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 µg/mL of lysine (p < 0.05) and 10 µg/mL histidine (p < 0.001), 100 µg/mL of glutamic acid (p < 0.05) and 200 µg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin is the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi

© Medina et al.Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

Acute administration of unacylated ghrelin has no effect on Basal or stimulated insulin secretion in healthy humans.

Unacylated ghrelin (UAG) is the predominant ghrelin isoform in the circulation. Despite its inability to activate the classical ghrelin receptor, preclinical studies suggest that UAG may promote β-cell function. We hypothesized that UAG would oppose the effects of acylated ghrelin (AG) on insulin secretion and glucose tolerance. AG (1 µg/kg/h), UAG (4 µg/kg/h), combined AG+UAG, or saline were infused to 17 healthy subjects (9 men and 8 women) on four occasions in randomized order. Ghrelin was infused for 30 min to achieve steady-state levels and continued through a 3-h intravenous glucose tolerance test. The acute insulin response to glucose (AIRg), insulin sensitivity index (SI), disposition index (DI), and intravenous glucose tolerance (kg) were compared for each subject during the four infusions. AG infusion raised fasting glucose levels but had no effect on fasting plasma insulin. Compared with the saline control, AG and AG+UAG both decreased AIRg, but UAG alone had no effect. SI did not differ among the treatments. AG, but not UAG, reduced DI and kg and increased plasma growth hormone. UAG did not alter growth hormone, cortisol, glucagon, or free fatty acid levels. UAG selectively decreased glucose and fructose consumption compared with the other treatments. In contrast to previous reports, acute administration of UAG does not have independent effects on glucose tolerance or β-cell function and neither augments nor antagonizes the effects of AG.

The Effects of Brominated Flame Retardants on Thyroid Hormone Homeostasis in Human Placenta Tissues and Cell Culture

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.

In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.

In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation.

In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure.

This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Glycogen Synthase Kinase 3 Influences Cell Motility and Chemotaxis by Regulating Phosphatidylinositol 3 Kinase Localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. It was initially found that comparing to wild type cells, gsk3- cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.

El fenotipo "basal-her2" en el cáncer de mama: caracterización clínico-molecular e implicaciones terapéuticas

Clinically HER2+ (cHER2+) breast cancer (BC), as exclusively determined by immunohistochemistry of HER2 protein overexpression and/or fluorescence in situ hybridization of HER2 gene amplification, has been largely considered a single disease entity in terms of clinical outcome and in the susceptibility to the anti-HER2 monoclonal antibody trastuzumab (Herceptin). However, although the adjuvant/neoadjuvant use of the trastuzumab has been shown to significantly reduce recurrence risk when added to standard chemotherapy in women with early-stage cHER2+ BC, not all cases derive similar benefit from trastuzumab because a significant number of cHER2+ BC patients develop disease recurrence. Unfortunately, the identification of a robust clinical predictor of trastuzumab benefit, including HER2 itself, has proven challenging in the adjuvant/neoadjuvant setting. Thus, we suggest that a new generation of research needs to refine the prognostic taxonomy of cHER2+ BC and develop easy-to-use, clinicbased prediction algorithms to distinguish between good- and poor- responders to trastuzumab-based therapy ab initio. This study offered two hypotheses: 1.) HER2 overexpression can unexpectedly take place in a molecular background owned by basal-like BC (a commonly HER2-negative BC subtype which possesses many epithelial-mesenchymal transition (EMT) characteristics and exhibits robust cancer stem cell [CSC]-like features), thus generating a so-called basal/cHER2+ BC subtype; 2.) the basal/cHER2+ phenotype confers poor prognosis and delineates a subgroup of intrinsically aggressive cHER2+ BC with primary resistance to trastuzumab...

Effective Cell-Centred Time-Domain Maxwell's Equations Numerical Solvers

This research work analyses techniques for implementing a cell-centred finite-volume time-domain (ccFV-TD) computational methodology for the purpose of studying microwave heating. Various state-of-the-art spatial and temporal discretisation methods employed to solve Maxwell's equations on multidimensional structured grid networks are investigated, and the dispersive and dissipative errors inherent in those techniques examined. Both staggered and unstaggered grid approaches are considered. Upwind schemes using a Riemann solver and intensity vector splitting are studied and evaluated. Staggered and unstaggered Leapfrog and Runge-Kutta time integration methods are analysed in terms of phase and amplitude error to identify which method is the most accurate and efficient for simulating microwave heating processes. The implementation and migration of typical electromagnetic boundary conditions. from staggered in space to cell-centred approaches also is deliberated. In particular, an existing perfectly matched layer absorbing boundary methodology is adapted to formulate a new cell-centred boundary implementation for the ccFV-TD solvers. Finally for microwave heating purposes, a comparison of analytical and numerical results for standard case studies in rectangular waveguides allows the accuracy of the developed methods to be assessed.