gamma-synuclein has a dynamic intracellular localization

gamma-Synuclein is a member of the synuclein family consisting of three proteins. Within the last several years increasing attention has focused on these proteins because of their role in human diseases. alpha-Synuclein relevance to Parkinson's disease is based on mutations found in familial cases of the disease and its presence in filaments and inclusion bodies in sporadic cases. gamma-Synuclein is implicated in some forms of cancer and ocular diseases, while beta-synuclein may antagonize their pathological functions. In this paper we present data on the localization and properties of gamma-synuclein in several neuronal and nonneuronal cell cultures. We show that contrary to the current opinion, gamma-synuclein is not an exclusively cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area and may be associated to centrosomes. On late steps of mitosis gamma-synuclein is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions a translocation of gamma-synuclein from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. gamma-Synuclein overexpression reduces neurite outgrowth in a greater extent then alpha-synuclein overexpression. These data support the view that gamma-synuclein may change its intracellular localization and associate with subcellular structures in response to intracellular signaling or stress.     

Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats

The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations of Ca2+ signaling in sensory neurons. The naturally occurring amino acid taurine functions as an osmolyte, antioxidant, Ca2+ modulator, inhibitory neurotransmitter, and analgesic such that its depletion in diabetes may predispose one to neuronal hyperexcitability and pain. This study reports the effects of taurine replacement on hyperalgesia and sensory neuron Ca2+ homeostasis in streptozotocin-diabetic (STZ-D) rats. Nondiabetic and STZ-D rats were treated with a 2% taurine-supplemented diet for 6-12 wk. Thermal hyperalgesia and mechanical allodynia were determined by measuring hindpaw withdrawal latency to radiant heat and the withdrawal threshold to the von Frey anesthesiometer. Intracellular Ca2+ signaling was explored in neurons from L4-L6 dorsal root ganglia (DRG), using fura 2 fluorescence. Taurine replacement of diabetic rats attenuated deficits of nerve conduction and prevented reductions of mechanical and thermal withdrawal threshold and latency, respectively. In small DRG sensory neurons from diabetic rats, recovery of intracellular Ca2+ concentration ([Ca2+]i) in response to KCl was slowed and 73% corrected by taurine. The amplitudes of caffeine and ATP-induced [Ca2+]i transients were decreased by 47 and 27% (P < 0.05), respectively, in diabetic rat DRG sensory neurons and corrected by 74 and 93% (P < 0.05), respectively, by taurine replacement. These data indicate that taurine is important in the regulation of neuronal Ca2+ signaling and that taurine deficiency may predispose one to nerve hyperexcitability and pain, complicating diabetes.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

Tranexamic acid suppresses the release of mitochondrial DNA,protects the endothelial monolayer and enhances oxidative phosphorylation

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Hair Mineral and Trace Element Content in Children with Down’s Syndrome

Biological Trace Element Research - Chronic exposure to lead causes disruption to energy production mechanisms and tissue damage, in particular through its binding to thiol groups and competition...     

Social correlates of stress in adult males of the great gerbil,Rhombomys opimus,in years of high and low population densities

The great gerbil, Rhombomys opimus, is the most social species in the Gerbillinae. The social structure consists of family groups that occupy isolated systems of burrows consisting of one breeding male, from one to seven females, and juveniles. During a year of peak density and one of density decline, we studied the influence of group size, group composition, local density, and distance to the nearest groups on fecal corticosterone and testosterone concentrations in breeding males. We also examined the relationship of hormone concentrations to the survival of males during the summer drought between the spring and the fall. We found that males differed in concentrations of steroid hormones. Concentrations of testosterone were lower whereas those of corticosterone tended to be higher in a year of high population densities compared with higher testosterone and lower corticosterone in a year with a lower density. This finding suggests that stress may be greater in higher densities because of increased social contact. Stepwise regression analysis revealed a positive and significant influence of the number of adult females in a family group on concentrations of fecal corticosterone and testosterone in adult males. Concentrations of corticosterone were also significantly higher in males that disappeared from family groups between the spring and the fall compared with males still alive in family groups in the fall. There was no change in concentrations of testosterone. These results suggest that social interactions within large family groups may be an important source of stress for adult males.     

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI ⁺] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI ⁺] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI⁺] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI ⁺].     

Proteomic Profiling of Triple-negative Breast Carcinomas in Combination With a Three-tier Orthogonal Technology Approach Identifies Mage-A4 as Potential Therapeutic Target in Estrogen Receptor Negative Breast Cancer

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)⁻ and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.Breast cancer, although a very heterogeneous disease, can be divided into three therapeutically relevant fundamental disease entities, simply based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (Her2)¹ status (i.e. ER⁺ and/or Her2⁺, and ER⁻Her2⁻), as the major currently available breast cancer therapeutic options are based on the ability to target these proteins. Hormone receptor positive and hormone receptor negative breast cancers are disease entities with distinct morphological, genetic and biological behavior (1). Hormone receptor negative tumors, which constitute ∼30% of primary breast cancers, tend to be high-grade, more frequently BRCA1 and TP53 mutated, and, more importantly, are not amenable to endocrine therapy. Her2 is amplified in ∼18–20% of breast cancers, and is more frequently observed in hormone receptor negative tumors. Her2 amplification is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. Her2 status is also predictive for several systemic therapies, particularly for agents that target Her2. The development of a humanized monoclonal antibody against Her2 (trastuzumab) has resulted in reduction of the risk of recurrence and mortality in patients with Her2 amplification (2, 3). Although trastuzumab is considered one of the most effective targeted therapies currently available in oncology, a significant number of patients with Her2-overexpressing breast cancer do not benefit from it (4, 5).Breast tumors that do not express ER, PgR, or Her2 (ER⁻ PgR⁻ Her2⁻), as determined by immunohistochemistry (IHC), are generally referred to as triple negative breast cancers (TNBCs), and they are not candidates for targeted therapies (endocrine therapy or trastuzumab). Although TNBCs account for a relatively small proportion of breast cancer cases (10–15%), they are responsible for a disproportionate number of breast cancer deaths. TNBC tumors form a recognizable prognostic group of breast cancer with aggressive behavior that currently lacks the benefit of available systemic therapy (6–8). Given the need to develop molecular criteria to reproducibly categorize molecular breast tumor subtypes at the protein level and the lack of targeted therapies available to treat patients bearing TNBCs, we have implemented a systematic proteomics approach to identify, characterize, and evaluate proteins present in triple-negative tumors that could constitute an appropriate therapeutic target for the clinical management of this group of patients. To this end, based on the analysis of 78 individual TNBC samples, we have established a large, cumulative, 2D-PAGE database of proteins expressed by TNBCs, including some that could be of potential therapeutic value. Comparison of this TNBC protein database with protein databases of other breast cancer subtypes previously established by our laboratory allowed us to single out a number of proteins preferentially expressed in TNBCs for which targeted therapeutics exist. In this report we further focused on the characterization of one such target, the cancer/testis antigen, melanoma-associated antigen 4 - Mage-A4.Cancer/testis antigens (CTAs) are expressed in a large variety of tumor types, whereas their expression in normal tissues is restricted to male germ cells, which are immune-privileged because of their lack of or low expression of human leukocyte antigen (HLA) molecules (9). Several studies have shown the existence of natural cellular and humoral responses against some CTAs, indicating that they are appropriate targets for vaccine-based cancer immunotherapy (10–12). So far, the use of CTAs in immunotherapeutic approaches to cancer treatment has been tested in more than 60 early phase clinical trials, with varying success, and a few candidate products have reached late-stage clinical trials. One such candidate vaccine, Astuprotimut-R (GSK-249553), a Mage-A3 antigen-specific cancer immunotherapeutic agent, is currently under clinical evaluation by GlaxoSmithKline in the largest-ever treatment trial in lung cancer, called MAGRIT (Mage-A3 as Adjuvant nonsmall cell lunG canceR ImmunoTherapy) (13).At present, CTAs comprise about 150 members, more than half of which are encoded by large, recently expanded families on chromosome X (14; see also CTDatabase at www.cta.lncc.br; last accessed 01.09.2012). These genes are organized into clusters and have undergone rapid evolution, possibly because of positive selection. The biological functions of CTAs are not fully understood, but emerging evidence suggest that they direct the proliferation, differentiation, and survival of human germ line cells and may have similar effect in cancer cells. Mage-A4 protein belongs to the Mage-A family of CT antigens. The Mage-A family is composed by 12 proteins (14, 15) and many members of the Mage-A family of CTAs have been associated with cancer, including breast cancer (14, 16, 17). However, past studies reported mostly on MAGE genes rather than protein expression, or on the expression of Mage protein families and not on any given specific protein.In this paper we describe the identification of Mage-A4 in breast tumor biopsies using 2D PAGE coupled with MS proteomics, and follow the protein localization from the tumor cells, to the tumor microenvironment, and to the serum of a patient. Using a three-tier orthogonal technology approach that combined 2D PAGE silver staining coupled with MS, with 2D Western blotting, and IHC, we showed that high level Mage-A4 expression in breast tumors occurs almost exclusively in the receptor negative disease (TNBC and Her2⁺ER⁻PgR⁻). The existence of immunotherapeutic approaches targeting MAGE protein family members (Mage-A4 specific or with broader specificity) and the fact that we were able to detect its presence in serum suggest novel management options for patients bearing Mage-A4 positive TNBCs and Her2⁺ER⁻PgR⁻ tumors.