Interaction with functional membrane proteins—A common mechanism of toxicity for lipophilic environmental chemicals?

• 1.1. The effects of several phenols, anilines and aliphatic alcohols on yeast plasma membrane H⁺-ATPase and purine transport system as well as on Na⁺, K⁺-ATPase and adenosine uptake by Chinese hamster ovary cells (CHO) were investigated.
• 2.2. In all cases an inhibition was observed, which could be correlated with the octanol/water partition coefficients of the substances tested, thus making quantitative structure-activity predictions possible.
• 3.3. The observed effects correlated well with the influence of the chemicals on cell growth.
• 4.4. The results suggest a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes.

     

Interaction with functional membrane proteins--a common mechanism of toxicity for lipophilic environmental chemicals?

1. The effects of several phenols, anilines and aliphatic alcohols on yeast plasma membrane H(+)-ATPase and purine transport system as well as on Na+, K(+)-ATPase and adenosine uptake by Chinese hamster ovary cells (CHO) were investigated. 2. In all cases an inhibition was observed, which could be correlated with the octanol/water partition coefficients of the substances tested, thus making quantitative structure-activity predictions possible. 3. The observed effects correlated well with the influence of the chemicals on cell growth. 4. The results suggest a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes.     

A common mechanism for posttranslational activation of plasma membrane receptors?

The process for posttranslational acquisition of ligand binding function is remarkably similar for three receptors with dissimilar structures, namely, the insulin, epidermal growth factor, and acetylcholine receptors. These receptors lack the ability to bind ligand immediately after translation, but slowly (t1/2 = 30-45 min) acquire this capacity while in the endoplasmic reticulum. This activation step occurs with similar kinetics for all three receptors and, in each case, required N-linked glycosylation. Several lines of evidence suggest a common mechanism for the acquisition of ligand binding function that involves the rearrangement of metastable disulfide bonds formed during or immediately after translation. This process precedes subunit assembly of both insulin and acetylcholine receptors, which also occurs in the endoplasmic reticulum. The posttranslational processing steps leading to the acquisition of ligand binding function may be an example of a more general process affecting cell surface proteins.     

A1 toxicity in yeast. A role for Mg?

We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae. The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions. Alterations to the PO4, Ca, or K concentration had little effect on toxicity. Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species. The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast. Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F. Fuhrmann, A. Rothstein [1968] Biochim Biophys Acta 163: 325-330). Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg. In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain. Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport. We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems.     

Maternal steroids and contaminants in common tern eggs: a mechanism of endocrine disruption?

We looked for evidence for the hypothesis that exposure of female birds to polychlorinated biphenyls (PCBs) results in alteration of blood steroid hormone concentrations and alters subsequent hormone transfer of steroids to eggs. Eggs of three-egg clutches were collected from a PCB-exposed common tern (Sterna hirundo) colony (Ram Island, Buzzards Bay, MA, USA) and from a relatively clean colony (Bodkin Island, Chesapeake Bay, MD, USA), and were analyzed for concentrations of organochlorine contaminants and steroid hormones (17beta-estradiol, 5alpha-dihydrotestosterone, testosterone and androstenedione). There was no relationship between total PCBs and steroid concentrations considering all eggs together, considering eggs of different laying order or considering differences between sequentially laid eggs in a clutch. Similarly, concentrations of di- and tri-chlorinated biphenyls and steroids in eggs were not related. The concentrations of PCBs, mercury and selenium were below estimated thresholds for toxicity to embryos. Maternal steroids, except estradiol, were present in yolk of all eggs, with increasing concentrations in the second and third eggs laid. Our data provided no evidence for a maternal toxicological event that might alter the amount of maternal steroid hormone transferred to eggs.     

Polyunsaturated fatty acids stimulate superoxide formation in tumor cells: A mechanism for specific cytotoxicity and a model for tumor necrosis factor?

Some neoplastic cell lines are readily killed when incubated in the presence of polyunsaturated fatty acids (PUFA). In an attempt to elucidate this phenomenon, we studied PUFA-driven superoxide (O2-) production by cultured NS-1 cells (murine lymphoid tumor cells). We find: (1) Even in the absence of added PUFA, NS-1 cells generate O2- (i.e., reduce nitroblue tetrazolium). (2) addition of PUFA increases O2- by greater than 50%. (3) Artificial loading of NS-1 cells with liposome encapsulated superoxide dismutase prevents the majority of spontaneous and PUFA-driven NBT reduction. We conclude that PUFA drives O2- generation by tumor cells, that this generation is largely intracellular, and that this phenomenon may help explain toxicity of PUFA for tumor cells.     

Acute regulation of 5′-AMP-activated protein kinase by long-chain fatty acid,glucose and insulin in rat primary adipocytes

Palmitate increased AMPK (5′-AMP-activated protein kinase) activity, glucose utilization and 2-DOG (2-deoxyglucose) transport in rat adipocytes. All three effects were blocked by the AMPK inhibitor Compound C, leading to the conclusion that in response to an increase in long-chain NEFA (non-esterified fatty acid) concentration AMPK mediated an enhancement of adipocyte glucose transport, thereby providing increased glycerol 3-phosphate for FA (fatty acid) esterification to TAG (triacylglycerol). Activation of AMPK in response to palmitate was not due to an increase in the adipocyte AMP:ATP ratio. Glucose decreased AMPK activity and effects of palmitate and glucose on AMPK activity were antagonistic. While insulin had no effect on basal AMPK activity insulin did decrease AMPK activity in the presence of palmitate and also decreased the percentage effectiveness of palmitate to increase the transport of 2-DOG. It is suggested that activation of adipocyte AMPK by NEFA, as well as decreasing the activity of hormone-sensitive lipase, could modulate adipose tissue dynamics by increasing FA esterification and, under certain circumstances, FA synthesis.     

Manganese deficiency and toxicity: are high or low dietary amounts of manganese cause for concern?

Manganese is an essential trace element that is required for the activity of several enzymes. Manganese is also quite toxic when ingested in large amounts, such as the inhalation of Mn-laden dust by miners. This review examines Mn intake by way of the food supply and poses the question: Is there reason to be concerned with Mn toxicity or deficiency in free-living populations in North America? Although much remains to be learned of the functions of Mn, at present there are only a few vaguely described cases of Mn deficiency in the medical literature. Given the heterogeneity of the North American food supply, it is difficult to see the possibility of more than greatly isolated and unique instances of Mn deficiency. However, low Mn-dependent superoxide dismutase activity may be associated with cancer susceptibility, and deserves further study. There may be reasons, however, to be concerned about Mn toxicity under some very specialized conditions. Increasing numbers of young people are adopting a vegetarian lifestyle which may greatly increase Mn intake. Iron deficiency may increase Mn absorption and further increase the body-burden of Mn, especially in vegetarians. Mn is eliminated primarily through the bile, and hepatic dysfunction could depress Mn excretion and further contribute to the body burden. Would such a combination of events predispose substantial numbers of people to chronic Mn toxicity? At present, there is no definite proof of this occurring, but given the state of knowledge at the present time, more studies with longer time-frames and more sensitive methods of analysis are needed.     

Overexpression and refolding of human Cyclin D3. A reliable method or not?

Cyclin D3 is a regulatory protein associated in a variety of human tumors. Despite several studies involving Cyclin D1 and D2, there are few articles that investigate the role of Cyclin D3. Therefore, this study aimed to produce and characterize Cyclin D3 using the Escherichia coli expression system and a protein refolding protocol. The anionic detergent SDS was used to solubilize the protein, then the solution were kept at 4 °C to precipitate SDS. After removing the precipitate by centrifugation, the supernatant was applied to the Ni-NTA column to purify His- tagged Cyclin D3. The recombinant protein shown to be refolded in a denaturation study by fluorescence spectroscopy at increasing concentrations of guanidine hydrochloride. The protein secondary structure, evaluated by circular dichroism, is composed of 39 % α helix and 15 % β-strands. In addition, the study aimed to validate the refolding protocol used to obtain Cyclin D3 from E. coli inclusion bodies.     

Formation of autophagosomes in the pinealocytes of the rat and Mongolian gerbil (Meriones unguiculatus): A lysosome wrapping mechanism?

Summary Autophagosome formation in rat and gerbil pinealocytes is described. It starts with the setting up of a tubular acid phosphatase-rich cisterna which gradually wraps around cytoplasmic areas to be catabolized. In light of obtained findings, it seems that the autophagosome formation in pinealocytes is a type of lysosome wrapping mechanism.     

New insights into the role of mast cells in autoimmunity: Evidence for a common mechanism of action?

Mast cells are classically considered innate immune cells that act as first responders in many microbial infections and have long been appreciated as potent contributors to allergic reactions. However, recent advances in the realm of autoimmunity have made it clear that these cells are also involved in the pathogenic responses that exacerbate disease. In the murine models of multiple sclerosis, rheumatoid arthritis and bullous pemphigoid, both the pathogenic role of mast cells and some of their mechanisms of action are shared. Similar to their role in infection and a subset of allergic responses, mast cells are required for the efficient recruitment of neutrophils to sites of inflammation. Although this mast cell-dependent neutrophil response is protective in infection settings, it is postulated that neutrophils promote local vascular permeability and facilitate the entry of inflammatory cells that enhance tissue destruction at target sites. However, there is still much to learn. There is little information regarding mechanisms of mast cell activation in disease. Nor is it known how many mast cell-derived mediators are relevant and whether interactions with other cells are implicated in these diseases including T cells, B cells and astrocytes. Here we review the current state of knowledge about mast cells in autoimmune disease. We also discuss findings regarding newly discovered mast cell actions and factors that modulate mast cell function. We speculate that much of this new information will ultimately contribute to a greater understanding of the full range of mast cell actions in autoimmunity. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Overexpression of protein kinase C ? improves retention and survival of transplanted mesenchymal stem cells in rat acute myocardial infarction

We assessed the effects of protein kinase C ɛ (PKCɛ) for improving stem cell therapy for acute myocardial infarction (AMI). Primary mesenchymal stem cells (MSCs) were harvested from rat bone marrow. PKCɛ-overexpressed MSCs and control MSCs were transplanted into infarct border zones in a rat AMI model. MSCs and PKCɛ distribution and expression of principal proteins involved in PKCɛ signaling through the stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway were analyzed by immunofluorescence and western blot 1 day after transplantation. Echocardiographic measurements and histologic studies were performed at 4 weeks after transplantation, and MSC survival, expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGFβ), cardiac troponin I (cTnI), von Willebrand factor (vWF), smooth muscle actin (SMA) and factor VIII and apoptosis in infarct border zones were assessed. Rat heart muscles retained more MSCs and SDF-1, CXCR4, PI3K and phosphorylated AKT increased with PKCɛ overexpression 1 day after transplantation. MSC survival and VEGF, bFGF, TGFβ, cTnI, vWF, SMA and factor VIII expression increased in animals with PKCɛ-overexpressed MSCs at 4 weeks after transplantation and cardiac dysfunction and remodeling improved. Infarct size and apoptosis decreased as well. Inhibitory actions of CXCR4 or PI3K partly attenuated the effects of PKCɛ. Activation of PKCɛ may improve retention, survival and differentiation of transplanted MSCs in myocardia. Augmentation of PKCɛ expression may enhance the therapeutic effects of stem cell therapy for AMI.Irreversible and widespread loss of myocardial cells and subsequent ventricular remodeling induced by acute myocardial infarction (AMI) is the main cause of chronic heart failure¹ and globally >17 million people died of ischemic heart diseases in 2008.² Stem cell-based regenerative therapy for AMI is encouraging with respect to preclinical^{3, 4} and clinical data,^{5, 6, 7, 8} and this may soon be a therapeutic modality for injury resulting from coronary artery disease. Two problems – poor homing of transplanted cells to injury sites, and poor cell survival – require resolution before transplantation therapy can be broadly effective. The stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis has an important role during migration, proliferation and survival of stem cells, but using this knowledge to improve homing and survival of therapeutic stem cells has not been successful.Previous studies^{9, 10, 11} suggest that protein kinase C ɛ (PKCɛ) is essential for signal transduction for ischemic cardioprotection, but whether it has an effect on stem cell retention and survival and what mechanism underlies this effect is uncertain. We know that SDF-1 increased significantly in mesenchymal stem cells (MSCs) after treatment with PKC activator and decreased after treatment with a PKCɛ inhibitor in preliminary experiments, and our latest work indicates that activating PKCɛ improves migration and paracrine function of MSCs in vitro.¹² Thus, we suggest that PKCɛ overexpression in transplanted bone marrow MSCs (BMMSCs) would improve retention and survival of MSC''s and improve cardiac function and remodeling in a rat AMI model.     

A common neuronal code for perceptual processes in visual cortex? Comparing choice and attentional correlates in V5/MT.

In the past two decades, sensory neuroscience has moved from describing response properties to external stimuli in cerebral cortex to establishing connections between neuronal activity and sensory perception. The seminal studies by Newsome, Movshon and colleagues in the awake behaving macaque firmly link single cells in extrastriate area V5/MT and perception of motion. A decade later, extrastriate visual cortex appears awash with neuronal correlates for many different perceptual tasks. Examples are attentional signals, choice signals for ambiguous images, correlates for binocular rivalry, stereo and shape perception, and so on. These diverse paradigms are aimed at elucidating the neuronal code for perceptual processes, but it has been little studied how they directly compare or even interact. In this paper, I explore to what degree the measured neuronal signals in V5/MT for choice and attentional paradigms might reflect a common neuronal mechanism for visual perception.