Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Extended genetic diversity of bovine viral diarrhea virus and frequency of genotypes and subtypes in cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5 \u2032 UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction

Low-dose oral imatinib in the treatment of systemic sclerosis interstitial lung disease unresponsive to cyclophosphamide: a phase II pilot study

Introduction: Pulmonary involvement represents a major cause of death of systemic sclerosis (SSc) patients. Recent data suggest that tyrosine kinase inhibitors, such as imatinib, may be a therapeutic option for SSc patients. However, preliminary published clinical trials were inconclusive about imatinib efficacy and showed side effects. The purpose of this study was to verify efficacy and tolerability of low-dose imatinib on interstitial lung disease in a cohort of SSc patients unresponsive to cyclophosphamide therapy.Methods: Thirty consecutive SSc patients with active pulmonary involvement, unresponsive to cyclophosphamide, were treated with imatinib 200 mg/day for 6 months followed by a 6-month follow-up. A "good response" was defined as an increase of forced vital capacity (FVC) by more of 15% and/or increase of diffusing capacity of carbon monoxide (DLCO) > 15% and PaO2 > 90% of initial value and high-resolution computed tomography (HRCT)-scan pattern unchanged or improved.Results: Twenty-six patients completed the study. Three patients died and one patient was lost to follow-up. Four patients (15.32%) had a good response, 7 worsened and 15 had a stabilized lung disease. Overall, 19 (73.07%) patients had an improved or stabilized lung disease. After a 6-month follow-up, 12 (54.5%) of the 22 patients showed an improved or stabilized lung disease.Conclusions: Lung function was stabilized in a large proportion of patients unresponsive to cyclophosphamide therapy and a beneficial outcome emerged from the analysis of HRCT lung scans. There was no significant improvement of skin involvement, and the low dose was well tolerated. These data provide useful suggestions to design future randomized clinical trials for SSc therapeutics

Production of Multiple Brain-Like Ganglioside Species Is Dispensable for Fas-Induced Apoptosis of Lymphoid Cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells

Concanavalin A/IFN-Gamma Triggers Autophagy-Related Necrotic Hepatocyte Death through IRGM1-Mediated Lysosomal Membrane Disruption

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ−/− mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Essential Nutrients for Bone Health and a Review of their Availability in the Average North American Diet

Osteoporosis and low bone mineral density affect millions of Americans. The majority of adults in North America have insufficient intake of vitamin D and calcium along with inadequate exercise. Physicians are aware that vitamin D, calcium and exercise are essential for maintenance of bone health. Physicians are less likely to be aware that dietary insufficiencies of magnesium, silicon, Vitamin K, and boron are also widely prevalent, and each of these essential nutrients is an important contributor to bone health. In addition, specific nutritional factors may improve calcium metabolism and bone formation. It is the authors’ opinion that nutritional supplements should attempt to provide ample, but not excessive, amounts of factors that are frequently insufficient in the typical American diet