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#20405221   2010/05/26 Save this To Up

The execution phase of autophagy associated PCD during insect metamorphosis.

During metamorphosis of Manduca sexta, involution of labial glands follows an autophagic pathway towards programmed cell death (PCD). We looked for evidence of both caspase dependent and independent pathways of PCD by assaying for caspases -1, -2, -3, and -6, proteasomal protease, and cathepsins B & L, using fluorogenic substrates and aldehyde and chloromethylketone inhibitors. The substrates FR-AMC and RR-AMC, preferentially degraded by cathepsins B and L, were the most rapidly degraded, increasing in rate as the gland involuted. Digestion of YVAD-AMC (preferential substrate for caspase-1) and DEVD-AMC (substrate for caspases-3 & -7) was barely detectable, less than 0.02% (on a per-unit-protein basis) of that seen in vertebrate embryos induced to undergo apoptosis. Cleavage of VDVAD-AFC (substrate for caspase -2) and VEID-AFC (substrate for caspase -6) was also assessed, but activity was negligible. Mitochondrial membrane permeabilization (MMP) and cytochrome c release were not detected. Exogenous caspase substrate, polyadenosyl ribose phosphorylase (PARP), is cleaved by labial gland extracts, but only at an acidic pH of 5.5-6.0, and into fragments different from those generated by caspases (confirmed by N-terminal sequencing). The cysteine protease inhibitor leupeptin inhibits PARP cleavage, but the caspase inhibitor DEVD-CHO does not. However, potential caspase-derived fragments of PARP are seen when cytochrome c and dATP are added to cytosolic extracts. Although apoptotic machinery is conserved and functional in this tissue, cell death occurs independently of caspases in metamorphosis. We also postulate that lysosomal proteases play the major proteolytic role similar to the caspase cascade seen in apoptosis.

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BACTERIOLOGY BACTEROIDES TCP-1 theta antibody Sour Recombinant Thermostable Recombinant Thermostable Recombinant Thermostable Recombinant Human PCDC5 P Recombinant Human PCDC5 P Recombinant Human PCDC5 P Recombinant Human PKC the Recombinant Human PKC the Recombinant Human PKC the Cell Meter™ Autophagy A

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#16106098   2005/08/17 Save this To Up

Release of IL-1beta via IL-1beta-converting enzyme in a skin dendritic cell line exposed to 2,4-dinitrofluorobenzene.

We used a mouse fetal skin dendritic cell line (FSDC) to study the effect of the strong allergen 2,4-dinitrofluorobenzene (DNFB) on interleukin (IL)-1beta release and IL-1beta receptor immunoreactivity. Stimulation with DNFB (30 minutes) increased IL-1 release without changing the mRNA levels of the protein. Furthermore, DNFB increased transiently the interleukin-1beta-converting enzyme (ICE) activity, as measured with its fluorogenic substrate Z-Tyr-Val-Ala-Asp-AFC. The ICE inhibitor Z-YVAD-FMK prevented the release of IL-1beta evoked by DNFB. Incubation of the cells with DNFB (30 minutes) strongly increased IL-1beta receptor immunoreactivity. The rapid effect of DNFB on the release of mature IL-1beta, without inducing an increase of IL-1beta mRNA in FSDC, suggests a posttranslational modification of pro-IL-1beta by ICE activity.

2943 related Products with: Release of IL-1beta via IL-1beta-converting enzyme in a skin dendritic cell line exposed to 2,4-dinitrofluorobenzene.

CELLKINES Natural Human I Human Interleukin-32 alph Human Interleukin-1-alpha Rat Anti-Mouse Interleuki Rat Anti-Mouse Interleuki Mouse Anti-Human Interleu Mouse Anti-Human Interleu Rat Anti-Mouse Interleuki Rat Anti-Mouse Interleuki Rat Anti-Mouse Interleuki Rat Anti-Mouse Interleuki Mouse Anti-Human Interleu

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#10706374   2000/04/13 Save this To Up

Changes of caspase activities involved in apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysaccharide (LPS) and cycloheximide.

The addition of lipopolysaccharide (LPS) together with cycloheximide (CHX) induced apoptosis in a subline of a J774.1 macrophage-like cell line, JA-4, as judged by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-staining and poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP)-cleavage. Caspase activities were examined in these macrophages in vitro using fluorogenic substrates such as acetyl-DEVD-aminomethyl coumarine (Ac-DEVD-AMC, caspase-3-like), acetyl-YVAD-aminomethyl coumarine (Ac-YVAD-AMC, caspase-1-like), acetyl-VEID-aminomethyl coumarine (Ac-VEID-AMC, caspase-6-like), and carbobenzoxy-IETD-aminofluoro coumarine (Z-IETD-AFC; caspase-8-like). Kinetic studies revealed these caspase activities with different Km and Vmax values in extracts of apoptotic macrophages. In the course of apoptosis, caspase-3-like activity increased first at 75 min, simultaneously with the appearance of TUNEL staining and prior to PARP cleavage, and then caspase-6 and 8-like activities increased at 90 and 105 min, respectively. However, caspase-1-like activity did not change throughout the experiment. Furthermore, removal of LPS and CHX by extensive washing of the cells for 60 min completely abolished the apoptosis and the subsequent release of lactate dehydrogenase (LDH) during additional incubation until 4 h after LPS addition. However, washing of the cells after 75 min or later resulted in the progress of apoptosis and LDH release, which was coordinated with the elevation of caspase-3-like activity at 60 min and that of caspase-6 or 8-like activity at 90 min, but not with that of caspase-1-like activity. These results suggest that caspase-3-like activity represents the most apical caspase among these caspases in terms of the intiation of apoptosis in macrophages treated with LPS and CHX. In the present study, we also provide evidence on the relatively low specificities of a series of caspase inhibitors other than acetyl-DEVD-aldehyde (Ac-DEVD-CHO) which specifically inhibited the caspase-3-like activity.

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Cell Meter™ Caspase 3 7 Cell Meter™ Caspase 3 7 Cell Meter™ Caspase 3 7 Cell Meter™ Caspase 8 A Cell Meter™ Caspase 9 A Cell Meter™ Caspase 9 A Cell Meter™ Caspase 9 A Cell Meter™ Caspase 8 A Cell Meter™ Caspase 9 A Cell Meter™ Annexin V B Cell Meter™ Annexin V B Cell Meter™ Annexin V B

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#10438906   1999/09/09 Save this To Up

Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

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voltage-dependent calcium Rabbit Anti-IEX1 Differen Rabbit Anti-IEX1 Differen Rabbit Anti-Apoptosis enh Rabbit Anti-Apoptosis enh anti-Caspase-2, Rabbit po anti-CAD (caspase-activat Anti AGO2 Human, Monoclon Anti AGO2 Human, Monoclon Human IgM antibody, Monoc Human IgG antibody, Monoc Human IgA antibody, Monoc

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#9620337   1998/06/30 Save this To Up

Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases.

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.

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Sterile filtered mouse s Anti AGO2 Mouse, Monoclon Anti AGO2 Mouse, Monoclon anti FAS IgG1 (monoclonal anti FAS IgM (monoclonal) HIV1 integrase antibody, Goat Anti-Mouse SAR1, (in Goat Anti-Mouse Rab17 (mo Goat Anti-Mouse IA2, (int Goat Anti-Human, Mouse HI Goat Anti-Human FTO (Mous Goat Anti-Human, Mouse EB

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#9590441   1998/06/23 Save this To Up

Activation of caspase-3 and apoptosis in cerebellar granule cells.

Caspase-3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal medium Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac-DEVD-amc, but not with Ac-YVAD-afc, a preferred substrate for caspase-1. This provided a basis to examine relationships between enzyme activity and cell viability for purposes of selecting an optimal time for comparing neuroprotective agents or strategies. Exposure of neurons to an apoptotic medium containing 5 mM K+ in absence of serum led to a rapid 5- to 10-fold increase in caspase-3 within 2-4 hr but without significant cell loss, or morphological alterations. Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 mM K+ and serum led to a rapid fall in caspase-3 and prevention of cell death. This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems for rapid enzyme turnover and/or activation of cytoplasmic components linked to later DNA degradation. The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase-3 dependent pathway for necrotic cell damage. Brief treatment with 10 microM LIGA20, an anti-necrotic agent, also attenuated cell loss and caspase-3 activity, indicating a broad spectrum of neuroprotection. Rapid and long-lasting effects, together with its biophysical properties, suggest that this semisynthetic ganglioside acted upstream at or near a membrane site. As such, gangliosides provide useful agents to further probe pathways relevant to neuronal death in culture.

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Interleukin-34 IL34 (N-t Interleukin-34 IL34 anti Sterile filtered mouse s anti HSV (II) gB IgG1 (mo anti HCMV IE pp65 IgG1 (m anti HCMV gB IgG1 (monocl DNA (cytosine 5) methyltr Human Interleukin-33 IL-3 Human Interleukin-32 alph Caspase-3 Inhibitor Z-DEV Caspase-3 Inhibitor Z-DEV Caspase 3 Inhibitor Z DEV

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