Altered Cellular Mechanisms Research Articles

Modeling SILAC Data to Assess Protein Turnover in a Cellular Model of Diabetic Nephropathy.

Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.

Neurodegenerative Disorders of Alzheimer, Parkinsonism, Amyotrophic Lateral Sclerosis and Multiple Sclerosis: An Early Diagnostic Approach for Precision Treatment.

Neurodegenerative diseases (NDs) are characterised by progressive dysfunction of synapses, neurons, glial cells and their networks. Neurodegenerative diseases can be classified according to primary clinical features (e.g., dementia, parkinsonism, or motor neuron disease), anatomic distribution of neurodegeneration (e.g., frontotemporal degenerations, extrapyramidal disorders, or spinocerebellar degenerations), or principal molecular abnormalities. The most common neurodegenerative disorders are amyloidosis, tauopathies, a-synucleinopathy, and TAR DNA-binding protein 43 (TDP-43) proteopathy. The protein abnormalities in these disorders have abnormal conformational properties along with altered cellular mechanisms, and they exhibit motor deficit, mitochondrial malfunction, dysfunctions in autophagic-lysosomal pathways, synaptic toxicity, and more emerging mechanisms such as the roles of stress granule pathways and liquid-phase transitions. Finally, for each ND, microglial cells have been reported to be implicated in neurodegeneration, in particular, because the microglial responses can shift from neuroprotective to a deleterious role. Growing experimental evidence suggests that abnormal protein conformers act as seed material for oligomerization, spreading from cell to cell through anatomically connected neuronal pathways, which may in part explain the specific anatomical patterns observed in brain autopsy sample. In this review, we mention the human pathology of select neurodegenerative disorders, focusing on how neurodegenerative disorders (i.e., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis) represent a great healthcare problem worldwide and are becoming prevalent because of the increasing aged population. Despite many studies have focused on their etiopathology, the exact cause of these diseases is still largely unknown and until now with the only available option of symptomatic treatments. In this review, we aim to report the systematic and clinically correlated potential biomarker candidates. Although future studies are necessary for their use in early detection and progression in humans affected by NDs, the promising results obtained by several groups leads us to this idea that biomarkers could be used to design a potential therapeutic approach and preclinical clinical trials for the treatments of NDs.

Involvement of 4-hydroxy-2-nonenal in the pathogenesis of pulmonary fibrosis.

Pulmonary fibrosis is a chronic progressive disease with high incidence, prevalence, and mortality rates worldwide. It is characterized by excessive accumulation of extracellular matrix in the lung parenchyma. The cellular and molecular mechanisms involved in its pathogenesis are complex, and some are still unknown. Several studies indicate that oxidative stress, characterized by overproduction of 4-hydroxy-2-nonenal (4-HNE), is an important player in pulmonary fibrosis. 4-HNE is a highly reactive compound derived from polyunsaturated fatty acids that can react with proteins, phospholipids, and nucleic acids. Thus, many of the altered cellular mechanisms that contribute to this disease can be explained by the participation of 4-HNE. Here, we summarize the current knowledge on the molecular states and signal transduction pathways that contribute to the pathogenesis of pulmonary fibrosis. Furthermore, we describe the participation of 4-HNE in various mechanisms involved in pulmonary fibrosis development, with a focus on the cell populations involved in the initiation, development, and maintenance of the fibrotic process, mainly alveolar cells, endothelial cells, macrophages, and inflammatory cells. Due to its characteristic activity as a second messenger, 4-HNE, in addition to being a consequence of oxidative stress, can support maintenance of the inflammatory and fibrotic process by spreading the effects of reactive oxygen species (ROS). Thus, regulation of 4-HNE levels could be a viable strategy to reduce its effects on the mechanisms involved in pulmonary fibrosis development.

Alcohol‐induced damage and death to developing and differentiated neurons.

Alcohol intoxication can induce foetal alcohol spectrum disorder after prenatal exposures, as well as damage neurons and contribute to neurodegeneration and cognitive decline in adults. The effect of alcohol on driving altered cellular mechanisms and post‐translational modification of proteins was investigated in both undifferentiated and differentiated human neural progenitor cells and SH‐SY5Y neuronal cells to mimic alcohol exposures during neuronal development and in adults. Human neural progenitor cells were differentiated into neuronal cells upon growth factor withdrawal, while all‐trans retinoic acid and brain‐derived nutrient factor were applied to differentiate SH‐SY5Y cells prior to a challenge with 0–200 mM alcohol for 3, 6, 12, or 24 hours. Alcohol exposure induced cell cytotoxicity in a concentration and time‐dependent fashion, with cell viability assessed using MTT, ATP, and LDH assays. Reactive oxygen species (ROS) generated in response to alcohol treatment were quantified using a dichlorofluorescin diacetate assay. The accumulation and quantitation of oxidatively damaged proteins was assessed by quantitation of protein carbonyl content and specific carbonyl group immuno‐blotting. In summary, at commonly encountered alcohol exposures, alcohol triggered cellular damage and death in developing as well as differentiated neurons.Support or Funding InformationCommonwealth Scholarship Commission

Recent Advances in the Realm of Allosteric Modulators for Opioid Receptors for Future Therapeutics.

Opioids, and more specifically μ-opioid receptor (MOR) agonists such as morphine, have long been clinically used as therapeutics for severe pain states but often come with serious side effects such as addiction and tolerance. Many studies have focused on bringing about analgesia from the MOR with attenuated side effects, but its underlying mechanism is not fully understood. Recently, focus has been geared toward the design and elucidation of the orthosteric site with ligands of various biological profiles and mixed subtype opioid activities and selectivities, but targeting the allosteric site is an area of increasing interest. It has been shown that allosteric modulators play key roles in influencing receptor function such as its tolerance to a ligand and affect downstream pathways. There has been a high variance of chemical structures that provide allosteric modulation at a given receptor, but recent studies and reviews tend to focus on the altered cellular mechanisms instead of providing a more rigorous description of the allosteric ligand's structure-function relationship. In this review, we aim to explore recent developments in the structural motifs that potentiate orthosteric binding and their influences on cellular pathways in an effort to present novel approaches to opioid therapeutic design.

Abstract B38: Splice variants in aggressive human breast cancer subtypes

Abstract Alternative splicing happens in all types of cells including benign or normal cells. However, the expression levels of the non-canonical variants in the normal cells are low that there is no notable effect by them. Studies have shown that altered cellular mechanisms in tumor cells can affect the transcription and splicing processes. If a variant is able to fold into a stable structure similar to that of the canonical variant, it may mimic the structural features and interact with interaction partners without processing them further. Thus, alternative splicing could provide a mechanism for turning an activator into an effective inhibitor. Moreover, the ratio of the expression of the splice isoforms plays a significant role in the normal functioning of a biological system. We hypothesize that due to the altered mechanisms in tumor environment, some non-canonical variants are expressed at higher levels that they have distinct functions compared to their corresponding canonical proteins or may even be drivers of certain pathological processes. In this study, we propose to analyze the alternative splice variants that are expressed in ERBB2+ (ERBB2+, ER-, PR-) subtypes of breast cancer. ERBB2+ subtypes are known to be aggressive with poor prognosis and account for 15-20% of breast cancers in US. Breast cancer cell lines have been used widely to investigate breast cancer mechanisms and to develop new therapeutic approaches. SKBR3, which is ER –, PR - with ERBB2/HER2 amplification has been used successfully as a preclinical model to screen for therapeutic agents targeting ERBB2 and to delineate mechanisms of resistance to ERBB2-based therapies. SUM190 and SUM149 serve as models for inflammatory breast cancer, the most lethal form of breast cancer. Both these cell lines are ER –, PR – and clinically very aggressive, but ERBB2 is amplified in SUM190 and expressed at low levels in SUM149. The contrasting ERBB2 expression observed in the similar clinical IBC phenotype represented by SUM149 and SUM190 and over-expression of ERBB2 in SKBR3, representing a non IBC epithelial adenocarcinoma tumor type, makes this group of cell lines ideal for the comparisons that we set out to produce. By integrating the proteomics and RNA-Seq data from SKBR3, SUM190 and SUM149, we studied the splice variants that are expressed in the Her2+ breast cancer subtypes. We identified more than one splice variant for 1904 genes expressed in at least one of the three cancer cell lines. Multiple variants of genes that are key players in the downstream ERBB signaling pathways along with variants specific to one cancer cell line compared to the other two cancer cell lines and normal mammary cells were found. The top-ranking Gene Ontology and BioCarta pathways for these specific variants pointed to distinct mechanisms; aminoglycan metabolism for SKBR3, lipid biosynthesis and metabolism in SUM190 and cell adhesion in SUM149. The transcript profiles for mRNA splicing mechanism show similarities between the inflammatory models SUM190 and SUM149, which was further supported by the identification of a novel peptide from the intronic region of 17-beta dehydrogenase 4 from both of these cell lines. The overall transcript profile and some key ERBB downstream pathways show a more robust clustering between SKBR3 and SUM190. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell lines may unveil their roles in the underlying cancer progression and metastasis. Citation Format: Rajasree Menon, Hogune Im, Michael Snyder, Emma (Yue) Zhang, Rui Chen, Shiaw-Lin Wu, William S. Hancock, Gilbert S. Omenn. Splice variants in aggressive human breast cancer subtypes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B38.

The cellular mechanisms of dry eye: From pathogenesis to treatment

Dry eye is a complex disease characterized by changes in the ocular surface epithelia related to reduced quality and/or quantity of tears, inflammatory reaction, and impairment of ocular surface sensitivity. It has recently been proposed that increased tear osmolarity represents a main trigger to the altered cellular mechanisms leading to epithelial damage in dry eye. However, dry eye pathogenesis is multifactorial, with cytotoxic inflammatory mediators, altered lacrimal gland secretion and nerve function, squamous metaplasia of the conjunctival epithelium and decrease of goblet cells density, all playing a role in a detrimental loop that perpetuates and worsens damage to the corneal and conjunctival epithelia. Current topical treatments for dry eye patients include the use of lubricants and anti-inflammatory drugs. However, lubricants only improve symptoms temporarily, and chronic use of topical steroids is associated to severe ocular side effects such as cataract and glaucoma. The deeper understanding of the cellular mechanisms that are altered in dry eye is opening novel perspectives for patients and physicians, who are seeking treatments capable not only of improving symptoms but also of restoring the homeostasis of the ocular surface. In this review, we will focus on novel anti-inflammatory agents and on nerve growth factor, a neurotrophin that is altered in dry eye and has been suggested as a main player in the neuroimmune cross-talk of the ocular surface as well as in the stimulation of corneal sensitivity, epithelial proliferation and differentiation, and stimulation of mucin production by goblet cells. J. Cell. Physiol. 228: 2253-2256, 2013. © 2013 Wiley Periodicals, Inc.

Thirteenth Annual Meeting of the Society for Pediatric Anesthesia, Dallas, Texas, October 8, 1999

Thirteenth Annual Meeting of the Society for Pediatric Anesthesia, Dallas, Texas, October 8, 1999

Mitogen stimulated rise of intracellular calcium concentration in single t lymphocytes from patients with major depression is reduced

1. 1. The authors investigated the signal transduction in T-lymphocytes as a peripheral model for central neurons. 2. 2. Intracellular free calcium concentration [Ca 2+] i was measured using fura 2 in T-lymphocytes from 6 patients with major depression during and after depression and from 6 healthy controls Patients were treated with interpersonal therapy (IPT) but not with psychotropic medication. 3. 3 Phytohemagglutinin (PHA) triggers an oscillatory [Ca 2+] i signal in human T-lymphocytes. This implies two mechanisms for [Ca 2+] i regulation: inositol phophate (IP) mediated release from intracellular stores and [Ca 2+] i influx from the extracellular medium. 4. 4. PHA stimulates 49% of T cells from controls but only 17% of T cells from depressed patients. This finding explains previous results from cells in suspension indicating that [Ca 2+] i signals after PHA-stimulation are reduced in cells from depressed patients. 5. 5 Cells from depressed patients show less [Ca 2+] i oscillations. Normal oscillation pattems are restored after clinical recovery from depression. 6. 6. Thus altered [Ca 2+] i oscillations in T-lymphocytes are a state phenomenon and may give us clues where to search for altered cellular mechanisms during depression.

The Aging Intervertebral Disk

This paper outlines the structure of the intervertebral disk and the process by which altered cellular mechanisms lead to deterioration with age. A reference list indicates current trends in research that attempt to explain the changing nature of the intervertebral disk and its role in spinal disease.