At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

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 flux 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, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

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

Immunological dysregulation is the cause of many non-infectious human diseases such as autoimmunity, allergy and cancer. The gastrointestinal tract is the primary site of interaction between the host immune system and microorganisms, both symbiotic and pathogenic. In this Review we discuss findings indicating that developmental aspects of the adaptive immune system are influenced by bacterial colonization of the gut. We also highlight the molecular pathways that mediate host–symbiont interactions that regulate proper immune function. Finally, we present recent evidence to support that disturbances in the bacterial microbiota result in dysregulation of adaptive immune cells, and this may underlie disorders such as inflammatory bowel disease. This raises the possibility that the mammalian immune system, which seems to be designed to control microorganisms, is in fact controlled by microorganisms.

The gut microbiota shapes intestinal immune responses during health and disease

It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.

CpG-rich islands and the function of DNA methylation

Human CD8 1 CXCR3 1 T cells have the same function as murine CD8 1 CD122 1 Treg

The host cells are protected from the indiscriminate attack of homologous complement by the membrane-associated complement regulatory proteins. A mouse monoclonal antibody (mAb) 512 (immunoglobulin Gi subclass) has recently been described that recognizes and inhibits the function of a rat complement regulatory protein, a rat homologue of mouse Crry/p65. The aim of this work is to assess the role of a complement regulatory protein (5l2Ag) recognized by mAb 512 in the complement-dependent glomerular injury induced by mAb 0X7 against rat Thy-1.1. For the induction of mesangial injury, the left kidney of a rat was perfused with a combination of 0X7 and 512 and the perfusate was discarded from the renal vein (Group I). After the renal artery and vein were restored, the left kidney was connected to the systemic circulation. Rats were euthanized 3 h, 2 days, and 14 days later. Rats perfused either with 0X7 (Group II) or with 512 (Group Ill) or with vehicle only (Group IV) were used as controls. At 3 h, rats of Group I showed more prominent cellular infiltration and mesangial lysis and more C3 deposition in the gbmeruli than rats of Group II. Rats of Groups Ill and IV showed no significant changes. At Day 2, there was still significant mesangial lysis and leukocyte infiltration in Group I rats, whereas rats in other groups showed an almost normal appearance. Glomerular

The Pole of a Complement Regulatory Protein in Rat Mesangial 2

Some R-mutant Escherichia coli and Salmonella heavily adhered to murine lymphoma cells of B cell and T cell lineages. This adhesion was primarily mediated by membrane-localized proteins on tumor cells, which bind the polymyxin B-reactive hydrophilic structure of lipis A on bacteria. SDS-PAGE analysis of tumor cell membranes showed that proteins or glycoproteins of MW = around 45Kd, 25-35Kd and around 15Kd preferentially bind lipid A. Various lymphoma cell lines binding the bacteria at different levels possessed lipid A-binding proteins of slightly different compositions. We conclude that lymphoma cells carry not a single but a group of lipid A-binding proteins in their membranes.

Demonstration of lipid A-binding proteins on murine lymphoma cells using R-mutant gram-negative bacteria as a detector.

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have great potentials for cell-based therapies based on their abundant potentials of self renewal and differentiation into all cell lineages (1,2) A serum-free ES cell differentiation system has an advantage for clinical applications because it can efficiently induce a specific cell lineage, and can avoid the risk of viral or prion infection by biomaterials This study was initiated to examine how to efficiently induce paraxial mesodermal progenitor cells from ES cells in serum-free cultures BMP4 acted as a key factor to promote the primitive streak-type mesoderm in both mouse development and ES cell differentiation in culture Many lateral mesodermal derivatives such as hematopoietic cells, endothelial cells and cardiomyocytes, and intermediate mesoderm derivatives such as renal progenitors have been induced by BMP4 stimulation However, differentiation of paraxial mesodermal cells from ES cells in serum-free culture has remained elusive In this study, we developed a simple culture system with BMP4 and lithium chloride (LiCl) in serum-free conditions to promote two types of paraxial mesodermal progenitors, osteochondrogenic progenitors, and myogenic progenitors, which were identified using the paraxial mesodermal marker PDGFR-α

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Specific Serum-free Conditions can Differentiate Mouse Embryonic Stem Cells into Osteochondrogenic and Myogenic Progenitors.

Neutrophils are key effector player of the innate immune system that are rapidly recruited to infected tissues to clear pathogens. Neutrophils also have the capacity to engulf damaged tissue cells to clear, although at the same time neutrophil granules including myeloperioxidase, elastase and ROS may damage tissues. We have shown previously that neutrophils help to repair wounded tissue. However, neutrophils are already differentiated cells, which cannot divide. For future personalized stem cells therapy, it is important to establish neutrophil-precursor cells, which will differentiate to mature neutrophils to engulf pathogens or damaged cells by the transplantation in vivo. First we characterized 32Dcl3 cell line, which has been established previously by WEHI-3 conditional medium. These cell lines differentiated to neutrophils by the stimulation of G-CSF. Then we established similar clone from C57BL/6 mice by culturing bone marrow cells in WEHI-3 conditional medium. We could obtain a neutrophil-precursor cell line (B6NPC), which grows more than six months in WEHI-3 conditional medium. By FACS analysis, we found that both 32Dcl3 and B6NPC had GR-1, CD11b and F4/80 cell surface markers, which are myeloid-lineage markers. However, these cells also have early T cell markers, which fit the hypothesis of myeloid-based model of hematopoietic cell differentiation. By the transplantation of B6NPC to syngeneic mice at the same time of wounding, we found early recovery from wound healing.

Ken-ichi Isobe

Papers

Human CD8 1 CXCR3 1 T cells have the same function as murine CD8 1 CD122 1 Treg

The Pole of a Complement Regulatory Protein in Rat Mesangial 2

Demonstration of lipid A-binding proteins on murine lymphoma cells using R-mutant gram-negative bacteria as a detector.

Specific Serum-free Conditions can Differentiate Mouse Embryonic Stem Cells into Osteochondrogenic and Myogenic Progenitors.

Establishment of neutrophil-lineage stem cells from C57BL/6 mice