Biomedical subjects

M L Shin

Publications and source records attributed to M L Shin.

At least 37 records · Page 2Linked to original sources

PubMed1994

Receptor-independent activation of guanine nucleotide-binding regulatory proteins by terminal complement complexes.

Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complement complexes (TCC) was investigated on human lymphoblastoid B-cell line JY25 and its mutant JY5 deficient in glycosylphosphatidylinositol-anchored proteins. TCC assembly achieved by antibody-dependent activation of C7-deficient serum reconstituted with C7 increased specific guanosine-5'-(gamma-thio)triphosphate (GTP gamma S) binding, 4- and 8-fold, in JY25 and JY5 membranes, respectively, between 2 and 10 min, over the level without C7. TCC also increased GTPase activity 5- and 4-fold in JY25 and JY5, respectively, between 5 and 10 min. Increased GTPase activity was noted first with C5b-7 assembly, which increased further with C5b-8 and C5b-9. The presence of G proteins in anti-TCC immunoprecipitates of cell lysates was investigated by demonstration of G alpha subunit that can be ADP-ribosylated by pertussis toxin (PTX). Immunoprecipitated TCC complexes contained a PTX-sensitive 41-kDa Gi alpha/Go alpha subunit, as shown by SDS-PAGE and Western blotting. These complexes were functionally active as determined by GTP gamma S binding. We have further shown that enhanced TCC elimination from the plasma membrane induced by TCC-generated signals was inhibited by PTX. In conclusion the biological activities induced by TCC in nucleated cells may be mediated in part by activation of PTX-sensitive G proteins.

B-Lymphocytes↗

PubMed1994

Ultrastructural studies of complement mediated cell death: a biological reaction model to plasma membrane injury.

Complement-mediated nucleated cell death has been shown to be independent of colloid-osmotic swelling. In contrast, other factors (e.g. Ca2+ influx) are of importance in the induction of cell death. In this communication, the sequential morphological features of complement-mediated cell injury have been studied by electron microscopy and compared with biochemical data (ATP content and LDH release). It was observed that immediately after C5b-8 lesion formation, although the overall cell, morphology is well preserved, the mitochondria display an "ultracondensed" appearance. Upon addition of C9, the mitochondria remain initially condensed, but swell progressively with final formation of flocculent densities. The nuclei become progressively edematous, with concurrent disappearance of heterochromatin. The nucleoli lose their associated chromatin and display segregation of their components with formation of markedly electron-dense filamentous deposits. The nuclear envelope remains initially intact, but subsequently progressive dilatation of the associated perinuclear RER cisterna and distention of the nuclear pores associated with leakage of chromatin into the cytoplasm are seen. The larger cell organelles (including mitochondria, ER, Golgi apparatus, etc.) become clustered around the nucleus, concurrently with marked edema of the outer cytoplasm and bleb formation. The RER cisternae become dilated, whereas the Golgi complex disappears. Relatively early on the plasma membrane shows breaks in continuity. The pattern of these changes--potentially related to Ca2+ influx, ATP efflux and overall metabolic depletion--corresponds to the previously described model of cell reaction to injury, confirming the dynamic nature of the process. The morphology of cell death in this model shares some features, e.g., the nucleolar changes, with "apoptosis" (programmed cell death). However, the overall pattern appears to correspond more to "necrosis," characterized by loss of volume control and mitochondrial abnormalities.

Adenosine Triphosphate↗

PubMed1994

Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack.

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.

Adenine Nucleotides↗

PubMed1994

Definition of a lipopolysaccharide-responsive element in the 5'-flanking regions of MuRantes and crg-2.

Macrophages are stimulated by lipopolysaccharide (LPS) of gram-negative organisms. The changes in LPS-stimulated macrophages include transcriptional activation of multiple immediate-early genes, which may contribute to the natural immunity to microorganisms. We have defined by deletion and mutational analysis LPS-responsive elements (LREs) in two chemokine genes, MuRantes and crg-2, which are activated in an immediate-early manner. LRE consists of two motifs, TCAYR, which is an AP-1 half site with two flanking bases, and (A/T) (G/C)NTTYC(A/T)NTTY, which resembles in part the interferon-stimulated responsive element (ISRE). The orientation of these two motifs relative to each other in MuRantes differed from that in crg-2. These two motifs are separated by 10 and 6 nonconsensus nucleotides in the MuRantes and crg-2 LREs, respectively. Stimulation of macrophage-like RAW 264.7 cells with alpha/beta interferon did not activate MuRantes, indicating that the ISRE-like motif in MuRantes does not have ISRE activity. Upon stimulation of RAW 264.7 cells with LPS, proteins capable of binding to LRE accumulate in the nuclei as measured by electrophoretic mobility shift assay. These LRE-binding proteins include c-Jun and CREB.

Animals↗

PubMed1993

Enhanced degradation of messenger RNA encoding myelin proteins by terminal complement complexes in oligodendrocytes.

Sublytic terminal C complexes (TCC) are capable of stimulating cells and affect the target cell activity. Activation of TCC that generates leukotriene B4 in oligodendrocytes, the myelin-forming cells of the central nervous system, is also a required process in antibody-mediated demyelination of rodent cerebellar explants. In the present study, the effect of TCC on myelin protein gene expression was studied in primary rat oligodendrocytes in culture. Sublytic activation of serum C reduced accumulation of mRNA encoding proteolipid protein (PLP) and myelin basic protein (MBP) within 1 h, but not beta-actin mRNA. C activation, on the other hand, induced sustained expression of c-jun mRNA. Experiments using C7-deficient human serum to determine the role of TCC showed that selective MBP and PLP mRNA down-regulation was achieved only when C7 was reconstituted to form TCC. The C7 requirement was also observed in the presence of alpha-amanitin. Post-transcriptional regulation was explored by determining mRNA decay, which demonstrated that the MBP and PLP mRNA were selectively destabilized when C7 was reconstituted. Limited exploration of the signals responsible for the TCC effect revealed that down-regulation of mRNA by TCC was significantly influenced by Ca2+ on PLP, whereas MBP did not show the same Ca2+ sensitivity as PLP. The TCC-mediated MBP mRNA decay was completely abrogated by HA1004, an inhibitor for the cAMP- and cGMP-dependent protein kinases, but not by H7, a protein kinase C inhibitor.

Amanitins↗

PubMed1993

Hydrolysis of myelin basic protein in myelin membranes by granzymes of large granular lymphocytes.

Immune-mediated demyelination initially manifests as a separation of myelin lamellae followed by loss of myelin proteins and eventual loss of myelin membranes. Myelin basic protein, one of the major structural proteins of myelin, is highly vulnerable to various proteases derived from diverse cell types. Killer lymphocytes such as CTL, in addition to other immune effectors, have been implicated in inflammatory demyelination. In addition to a pore forming peptide, the granules of CTL and large granular lymphocytes (LGL) contain a number of serine esterases collectively called as granzymes. We studied the effect of these granule enzymes on myelin and found that LGL granule-extracts (LGL-g) hydrolyzed MBP in myelin membranes. LGL-g also cleaved purified MBP at neutral pH in a Ca2+ independent manner and this hydrolysis was inhibited by serine esterase inhibitors. In addition, absorption of LGL-g with antigranzyme A significantly reduced MBP hydrolysis. These findings implicated the granzymes in LGL-g, especially granzyme A, as the agent causing MBP degradation. Inasmuch as activation of CTL in the vicinity of myelinated axons can lead to granule exocytosis, hydrolysis of MBP by granzymes may be a significant event in myelin destruction.

Cell Membrane↗

PubMed1993

Generation of diacylglycerol and ceramide during homologous complement activation.

Formation of sublytic terminal complement complexes (TCC) on nucleated cells produces transient increase in [Ca2+]i and activates protein kinase C. The present study is to evaluate whether TCC can generate endogenous signal messengers other than Ca2+ that regulate cell activities by measuring mass-levels of sn-1,2-diacylglycerol (DAG) and ceramide. As targets, lymphoblastoid human B cell lines JY25 and its mutant JY5 were used. JY5, cells deficient in glycosylphosphatidylinositol-anchored proteins with higher lytic susceptibility to human complement, are four times more efficient in forming C5b-9. When cells sensitized with limited anti-class II IgG were exposed to human serum to generate sublytic TCC, a sustained increase in DAG and ceramide was observed with a maximum 3.6-fold DAG increase over basal level in JY25 and 2.8-fold in JY5, and 6.3-fold ceramide increase in JY25 and 2.8-fold in JY5. The effect of TCC was evaluated with C7-deficient human serum (C7D) +/- C7 and also with C5b6, C7, C8, and C9 proteins. The DAG and ceramide increase by C7D + C7 over C7D control were 1.6- and 1.8-fold, respectively, in JY25, and 2.3-, and two-fold in JY5. TCC activation also induced an increased hydrolysis of sphyingomyelin and phosphatidylcholine. In addition, DAG increase by TCC was primarily achieved by C5b-7 and preincubation of cells with pertussis toxininhibited DAG increase, suggesting an involvement of a pertussis toxin-sensitive GTP-binding protein. As important signal transduction molecules, DAG and ceramide generated in response to TCC assembly, could participate in cell activation during inflammation and repair.

Animals↗

PubMed1993

Activation of the alternative complement pathway and production of factor H by skeletal myotubes.

Skeletal muscle myotubes from neonatal rats were used to study the interaction of skeletal muscle with complement. Serum from guinea pig, rabbit, and human, in the absence of muscle-specific antibody, caused creatine phosphokinase release, which required activation of the terminal complement cascade. Cleavage of serum C3 and Factor B in the presence of myotubes was dependent on Mg2+, but not Ca2+, and C3 cleavage occurred only in the presence of Factor B. Rat myotubes caused significant consumption of C8 and C9 in rat serum, which also required Mg2+, but not Ca2+. All of these findings are typical of a tissue capable of activating the alternative pathway. In addition, the C2 myotube cell line was shown to produce Factor H, an inhibitory protein of the alternative pathway, as demonstrated by Factor H mRNA expression and immunoprecipitation of the protein.

Animals↗

PubMed1992

Glutamate differentially inhibits the expression of class II MHC antigens on astrocytes and microglia.

MHC molecules are required for Ag recognition by T cells. Inasmuch as cells in the central nervous system do not express MHC constitutively, appearance of MHC, in inflammatory and degenerative diseases of the brain, may indicate local Ag presentation and subsequent immune response. Although both astrocytes and microglia are capable of class II MHC expression in vitro, in vivo studies failed to show the presence of significant amounts of class II on astrocytes compared to microglia. Our study is designed to clarify possible regulatory mechanisms that can explain the differences in inducibility of class II MHC between astrocytes and microglia in vivo. Using dissociated rat brain cell cultures, we have found that glutamate, an excitatory neurotransmitter, exerted a profound inhibitory effect on IFN-gamma-induced expression of class II on astrocytes, but not on microglia. Both glutamate and norepinephrine, a neurotransmitter previously reported to down-regulate class II on astrocytes, inhibited the induction of class II on astrocytes by eliminating accumulation of class II MHC mRNA. The kinetics of class II mRNA induction by IFN-gamma in the presence of glutamate suggested that glutamate may act as a transcriptional inhibitor. It is likely that class II induction on astrocytes in vivo may be selectively down-regulated by neurotransmitters such as glutamate and norepinephrine.

Animals↗

PubMed1992

Poly(A) removal is the kinase-regulated step in tumor necrosis factor mRNA decay.

Tumor necrosis factor (TNF) is a pleiotropic biomodulator and an important inducer of certain pathophysiologic immune reactions such as granuloma formation, cachexia, and septic shock. The production of TNF by astrocytes, which may figure prominently in the development of immune responses within the central nervous system, is subject to post-transcriptional regulation. We have previously shown that in virus-stimulated astrocytes, inhibition of protein kinase C results in a specific, 10-fold decrease in TNF mRNA half-life. Here we show that the decay of TNF messages induced in the macrophage-like cell line RAW 264.7 by either virus or lipopolysaccharide was subject to similar regulation, and that this pathway influenced the amount of TNF protein released by stimulated cells. Using a modified RNase protection assay, we demonstrate that inhibition of protein kinase C significantly enhanced the rate of poly(A) removal from TNF mRNA, thus facilitating an early event in the process of mRNA degradation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus.

Synthesis of complement proteins and their regulation in resident cells of the central nervous system are important pathophysiologic factors that can affect the outcome of inflammatory central nervous system diseases. Primary cultures of rat astrocytes constitutively express C3 mRNA and produce C3 protein; both of them were enhanced by LPS or by a live as well as inactivated Newcastle disease virus, a neurotropic paramixovirus. TNF, IL-1 beta, and IL-8 also increased the levels of C3 mRNA and protein whereas IL-1 alpha and IL-6 had no effect, although all of these cytokines are inducible by LPS. LPS stimulation in the presence of cycloheximide decreased the LPS-mediated C3 mRNA induction by 60%. These data suggest that LPS effect on C3 regulation is mediated directly by LPS as well as by LPS-induced cytokines. Interestingly, C3 mRNA induced by Newcastle disease virus or inactivated Newcastle disease virus was inhibited by protein kinase inhibitors, H-7 and staurosporine, whereas these inhibitors had no effect on C3 induction mediated by LPS or cytokines, indicating the existence of different signal transduction pathways.

Alkaloids↗

PubMed1992

Absence of measles, mumps, and rubella viral genomic sequences from multiple sclerosis brain tissue by polymerase chain reaction.

We tested for measles, mumps, and rubella viruses in multiple sclerosis by polymerase chain reaction (PCR). Using RNA extracted from 19 multiple sclerosis and 8 control brain specimens, nested PCR was performed after reverse transcription (RT) of the RNA to cDNA using primer pairs directed against two regions in the genomes of measles and mumps viruses and one region in the rubella virus genome. Despite enhanced sensitivity of nested RT PCR, measles, mumps, and rubella viral genomic sequences were not found in any brain specimen.

Adult↗

PubMed1991

Quantitative analysis of adenine nucleotides during the prelytic phase of cell death mediated by C5b-9.

The nucleated cell death mediated by C5b-9 depends on the extent of C fixation and parameters that affect the ability of the cell to eliminate C5b-9. When C5b-9 formation exceeds elimination, cell death can be initiated. High Ca2+ in the medium accelerates Ehrlich ascites cell death induced by a large number of C5b-9, whereas osmotic prevention of cell swelling has little effect in protecting Ehrlich cells from killing by C5b-9. In the present study, we investigated the interrelationship between intracellular Ca2+, intra- and extracellular adenine nucleotides, and mitochondrial membrane potential, to understand the mechanism of acute cell death induced by C5b-9. When Ehrlich cells carrying C5b-8 were exposed to C9, rapid and profound ATP depletion in the cell was observed before cell death. Leakage of the adenine nucleotides ATP, ADP, and AMP also began during the prelytic phase. Studies using digital imaging fluorescence microscopy showed that loss of mitochondrial membrane potential was noted immediately after C9 addition but before nuclear staining with propidium iodide. These findings suggest that an increase in intracellular Ca2+ through C5b-9 channels and loss of mitochondrial membrane potential may initiate rapid cell death. The prelytic leakage of ATP precursors may also contribute to cell death by decreasing nucleotide pools, because recovery of ATP production was observed after a similar degree of ATP loss in cells exposed to sublethal doses of KCN, in which ADP and AMP leakage was not present.

Adenine Nucleotides↗

PubMed1991

Inhibitors of membrane lipid metabolism enhance complement-mediated nucleated cell killing through distinct mechanisms.

The ability of nucleated cells to survive limited complement attack has been attributed to metabolic processes unique to these cells, such as rapid elimination of terminal complement complexes (TCC) from their surfaces. The biochemical processes activated by complement channels responsible for cell defense remain poorly defined. Metabolic inhibitors affecting membrane lipid turnover have been shown to increase the complement-mediated cell death. Whether these metabolic inhibitors increase lytic susceptibility of target cells by reducing the rate of TCC elimination has not been previously evaluated. In the present study, inhibitors of membrane lipid transmethylation and lysolecithin reacylation were evaluated in view of the observations that TCC concurrently increase lipid transmethylation and inhibit lysolecithin reacylation, and the inhibition of lipid transmethylation correlates with increased complement-mediated cell death. We have measured the formation as well as the elimination of C5b-9 on the target membrane that affect the outcome of cell death. Our results in the present communication indicated that inhibitors of transmethylation and lysolecithin reacylation increased TCC-mediated cell death through distinct pathways, the former by allowing more efficient deposition of TCC, and the latter by impairing TCC elimination.

1-Acylglycerophosphocholine O-Acyltransferase↗

PubMed1990

Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes.

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Multiple signal messengers generated by terminal complement complexes and their role in terminal complement complex elimination.

The best established function of C5b-9 is the ability to lyse or kill cells after assembly in the plasma membrane. In addition to this cytolytic function, increasing evidence suggests that C5b-9 also stimulate a variety of cell functions in vitro. Relatively little is known about the C5b-9 signals responsible for cell activation other than a transient increase in cytosolic Ca2+ primarily due to Ca2+ influx that have been determined in a cell population. In this report, signal messenger generation in Ehrlich cells by the sublytic terminal complement complexes (TCC), C5b-9, C5b-8, and C5b-7, was further examined, as well as the role of signal messengers in stimulating elimination of TCC from the cell surface. Changes in cytosolic Ca2+ were monitored in individual cells after a single dose of C5b-9 by digital imaging fluorescence microscopy that revealed oscillations in cytosolic Ca2+ over a period of 10 min. Sublytic C5b-9 substantially increased protein kinase C (PKC) activity at an external Ca2+ concentration of 1.5 mM. C5b-9-mediated PKC activation could be inhibited by 60 to 80% when external Ca2+ was reduced to 0.015 mM. C5b-8, but not C5b-7, activated PKC to a lesser extent. C5b-8 and C5b-7 also stimulated an increase in cAMP. Rapid elimination of TCC known to be stimulated by Ca2+ signal was partially inhibited by protein kinase inhibitors, H-7 and to a lesser extent by HA1004, suggesting a role for PKC in the elimination response. TCC elimination was not accelerated by agents that increase cAMP.

Calcium↗

PubMed1989

Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Distinct restriction of complement- and cell-mediated lysis.

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.

Antibody-Dependent Cell Cytotoxicity↗