Phosphatidylserine-mediated phagocytosis of anticancer drug-treated cells by macrophages

金沢大学医薬保健研究域薬学系Apoptotic cells are rapidly phagocytosed and eliminated from the organism. Although cancer cells apoptose when treated with anticancer drugs, how those cells are recognized by phagocytic cells has remained unclear. The human leukemia cell line Jurkat was cultured with doxorubicin or bufalin and induced to undergo apoptosis accompanied by phosphatidylserine externalization. When apoptotic Jurkat cells were mixed with mouse peritoneal macrophages, efficient phagocytosis was observed. Apoptosis and phagocytosis of Jurkat cells were both inhibited by Z-VAD-FMK, and phagocytosis was significantly reduced in the presence of phosphatidylserine-containing liposomes. These results suggest that anticancer drugs induce apoptosis- dependent and phosphatidylserine-mediated phagocytosis in cancer cells

Fruit fly as a model organism in the study of human diseases and drug discovery

Drug discovery is a dynamic yet an ever-lasting topic in medicine that heavily relies on the application of modelorganisms. Through the use of suitable model organisms, pre-clinical testing of new drug candidates can be carried out tensively prior to further testing with humans. This has been proven, so far, as one of efficient ways to limit the emergence of new substances that are potentially harmful to individuals. Nevertheless, increasing public interest in the thical issues raised by the use of live model organisms, such as mice and rats, pushes urgent needs for alternative modelorganisms. To this end, fruit fly Drosophila melanogaster might be an appropriate model organism to be accounted r.With its long-standing history of use, Drosophila is an insect behind the revelation of striking similarity to humans as to basic biological mechanisms and their tight controls to maintain homeostasis. Impairment of such events in rosophila is linked to the emergence of metabolic-related diseases such as obesity and diabetes mellitus, the unsolved cases of neurodegenerative diseases, and the fall of host immune responses against various infectious agents. With a igh genetic similarity, about 75%, to humans, ease of maintenance, and the availability of various disease models constructed through genetic manipulation and/or chemical induction, Drosophila has been a very promising model organism in he field of drug discovery. With this in mind, it would be not surprising if this tiny yet powerful model organism will soon substitute our current in vivo platform in the pre-clinical testing of new drug candidates

Phagocytic removal of cells that have become unwanted: Implications for animal development and tissue homeostasis

金沢大学医薬保健研究域薬学系Cells that have become unwanted need to be promptly, selectively, and safely removed. This is made possible by apoptosis-dependent phagocytosis, in which cells unnecessary, obstructive, or dangerous to organisms are induced to undergo apoptosis so that they are earmarked for phagocytosis. The phagocytic elimination occurs so quickly that cells with hallmarks of apoptosis are barely detectable in vivo. The removal of particular types of cells at appropriate stages of development not only contributes to the disposal of spent cells, the creation of space for morphogenesis, and the exclusion of pathogenic or noxious cells, but seems to actively control tissue renewal, tissue remodeling, tissue function, and pathogenic state. This event thus plays an indispensable role in the maintenance of animal development and tissue homeostasis. © 2011 The Authors. Journal compilation © 2011 Japanese Society of Developmental Biologists

Independence of plasma membrane blebbing from other biochemical and biological characteristics of apoptotic cells

金沢大学医薬保健研究域薬学系Plasma membrane blebs are observed in many types of apoptotic cells, but their physiological roles remain to be clarified. We examined whether there is a causative connection between membrane blebbing and other apoptotic changes in Jurkat cells induced to undergo apoptosis by doxorubicin in the presence or absence of Y-27632, an inhibitor of the Rho kinase ROCK-I. The inclusion of the drug made most membrane blebs disappear, while other changes, such as chromatin condensation, inactivation of mitochondrial enzymes, externalization of the membrane phospholipid phosphatidylserine, and removal of cell surface sialic acid, remained unaffected. Furthermore, these apoptotic cells were phagocytosed by macrophages as efficiently as normally apoptosing cells. These results indicate that blebbing of the plasma membrane occurs independently from other apoptotic changes and is not involved in the recognition and engulfment of apoptotic cells by macrophages

Multiplex PCRを用いた簡便で感度の高い溺死診断法の開発

For diagnosing death due to drowning, the method of acid digestion of diatoms is widely used to detect plankton in the organs of the corpse. However, the method is limited by its　being complex, hazardous, time-consuming, and insufficiently sensitive. We therefore, developed a novel simple method to diagnose death due to drowning, and determined the location of drowning by detecting genes of representative bacteria in the environment. To procure all the information in one step, the multiplex PCR method was designed. For the diagnosis of drowning, the genes of upper respiratory indigenous bacteria, Streptococcus salivarius and Streptococcus sanguinis were used as indicators. For detection of the location of drowning, Aeromonas hydrophila and Microcystis aeruginosa were used as indicators of freshwater, and Vibrio harveyi as an indicator of seawater. A set of primers was designed for multiplex PCR. to amplify all the bacterial genes simultaneously. Using this method, 47 cases of drowning were examined, and the causes and locations of death were diagnosed.博士（医学）・乙第1428号・平成31年3月15

Mechanisms and consequences of phagocytosis on influenza virus-infected cells

金沢大学医薬保健研究域薬学系Influenza virus-infected cells are induced to undergo apoptosis and become susceptible to phagocytosis. Data from our in vitro and in vivo experiments have suggested that 1) alveolar macrophages and neutrophils phagocytose influenza virus-infected cells in an apoptosis-dependent manner; 2) the membrane phospholipid phosphatidylserine and viral neuraminidase-processed carbohydrates at the surface of target cells and phagocytes, respectively, are involved in the association of the two types of cells; and 3) phagocytic elimination of virus-infected cells leads to a reduction in the pathogenesis of influenza. These findings could lead to the development of a novel antiviral agent against influenza. © 2008 Bentham Science Publishers Ltd.全文公開20090

口腔内所見を用いた新たな年齢推定法

In forensic science, age estimation is an important step in identifying unidentified cadavers. As teeth are resistant to environmental degradation for long periods of time, they are often used to estimate age. Although there have been many reports of age estimation based on dental morphology, these methods tend to be subjective and cannot be used i n the case of edentulous jaws. In the present study, we developed a new method of age estimation from dental parameters (number of upper teeth [UT], lower teeth [LT], and prostheses [NP]; tooth attrition [TA]; and occlusal area [OA]) and the mandibular angle (MA) measured at proposed an equation for calculating the age. The results show that the mean error of this method is similar to that of previous methods, and even demonstrated improved accuracy in subjects aged >60 years. We also proposed an equation for age estimation from only the MA, and showed that we can perform age estimation even in edentulous cases using this equation. Because our method is superior in its simplicity, objectivity, and applicability when compared with previous methods, we believe our method wll prove useful for age estimation in a wide variety of cases.博士（医学）・甲611号・平成26年3月17

A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis

金沢大学医薬保健研究域薬学系The structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens

Structural change of ribosomes during apoptosis: Degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

金沢大学医薬保健研究域薬学系Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, PO, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

金沢大学医薬保健研究域薬学系To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity. © 2005 The Japanese Biochemical Society