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D C Huang

Publications and source records attributed to D C Huang.

At least 19 recordsLinked to original sources
PubMed2001

Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis.

Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Gene structure alternative splicing, and chromosomal localization of pro-apoptotic Bcl-2 relative Bim.

Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5' to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases.

Alternative Splicing↗
PubMed2001

Tissue expression and subcellular localization of the pro-survival molecule Bcl-w.

Anti-apoptotic members of the Bcl-2 family, such as Bcl-w, maintain cell viability by preventing the activation of the cell death effectors, the caspases. Gene targeting experiments in mice have demonstrated that Bcl-w is required for spermatogenesis and for survival of damaged epithelial cells in the gut. Bcl-w is, however, dispensable for physiological cell death in other tissues. Here we report on the analysis of Bcl-w protein expression using a panel of novel monoclonal antibodies. Bcl-w is found in a diverse range of tissues including colon, brain and testes. A survey of transformed cell lines and purified hematopoietic cells demonstrated that Bcl-w is expressed in cells of myeloid, lymphoid and epithelial origin. Subcellular fractionation and confocal laser scanning microscopy demonstrated that Bcl-w protein is associated with intracellular membranes. The implications of these results are discussed in the context of the phenotype of Bcl-w-null mice and recent data that suggest that Bcl-w may play a role in colon carcinogenesis.

Animals↗
PubMed2001

Rapid selection against truncation mutants in yeast reverse two-hybrid screens.

The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner. Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells. A laborious component of this system is to eliminate those mutations that cause protein truncation. By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.

Apoptosis Regulatory Proteins↗
PubMed2000

Fas ligand-induced c-Jun kinase activation in lymphoid cells requires extensive receptor aggregation but is independent of DAXX, and Fas-mediated cell death does not involve DAXX, RIP, or RAIDD.

Jun kinase signaling can be elicited by death receptor activation, but the mechanism and significance of this event are still unclear. It has been reported that cross-linking Abs to Fas trigger c-Jun N-terminal kinase (JNK) signaling via caspase-mediated activation of MEKK1 (JNK kinase kinase), elevation of ceramide levels or by recruitment of death domain associated protein (DAXX) to Fas. The effect of physiological ligand for Fas on JNK signaling was never investigated, although evidence is accumulating that Fas ligand is able to induce cellular responses distinct from those evoked by Ab-mediated cross-linking of Fas. Therefore, we investigated the effect of Fas ligand on JNK signaling. Like its ability to induce cell death, Fas ligand reliably activated JNK only upon extensive aggregation of the receptor. Although this was partially dependent on caspase activation, DAXX was not required. DAXX and other death receptor-associated proteins, which have been reported to bind directly or indirectly to Fas, such as receptor interacting protein (RIP) and RIP-associated ICH-1/CED-3-homologous protein with a death domain (RAIDD), were shown to be dispensable for Fas ligand-induced apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Increased immunoreactive labeling of the spinal N-methyl-D-aspartate R1 receptors after dorsal root ganglionectomy in the rats.

The N-methyl-D-aspartate (NMDA) receptor plays an important role in the development of the autotomy after dorsal root ganglionectomy (DRGn). In this study, we further investigated the expression of the NMDAR1 in the spinal cord of the rats after right DRGn by immunohistochemical analyses. Computerized densitometric analysis of the NMDAR1 immunoreactivity was done and the integrated optical density (IOD) of the superficial laminae of the dorsal horn of the spinal cord was measured. The immunoreactive labeling of the NMDAR1 was increased in the cervical spinal cord ipsilateral to the DRGn from day 5 to 14 after DRGn. The ratio of the right/left IOD of the rats receiving DRGn was significantly higher than the rats in the sham-operated group and the control group (P<0.05). The expression of the NMDAR1 increased gradually to reach the peak at day 7 after DRGn (mean right/left IOD ratio=1.52), then decreased thereafter. The increased expression of the NMDAR1 at day 7 was suppressed by MK-801 (NMDA receptor antagonist) administered immediately after DRGn, but not by normal saline or 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX, non-NMDA receptor antagonist). The results indicated that the expression of the NMDAR1 in the superficial laminae of the dorsal horn of the spinal cord was increased after DRGn and the time course was compatible with the onset and development of the autotomy induced by DRGn.

Animals↗
PubMed2000

Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L).

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

Animals↗
PubMed2000

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

Amino Acid Sequence↗
PubMed2000

The proapoptotic BH3-only protein bim is expressed in hematopoietic, epithelial, neuronal, and germ cells.

Proapoptotic Bcl-2 family members activate cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. Bim belongs to a distinct subgroup of proapoptotic proteins that only resemble other Bcl-2 family members within the short BH3 domain. Gene targeting experiments in mice have shown that Bim is essential for the execution of some but not all apoptotic stimuli, for hematopoietic cell homeostasis, and as a barrier against autoimmunity. There are three Bim isoforms, Bim(S), Bim(L), and Bim(EL), which have different proapoptotic potencies due at least in part to differences in interaction with the dynein motor complex. The expression pattern of Bim was investigated by immunohistochemical staining, immunoprecipitation followed by Western blotting, and in situ hybridization. Bim was found in hematopoietic, epithelial, neuronal, and germ cells. Bim(L) and Bim(EL) were coexpressed at similar levels in many cell types, but Bim(S) was not detected. Microscopic examination revealed a punctate pattern of Bim(L) and Bim(EL) immunostaining, indicating association with cytoplasmic structures. These results are discussed in the context of the phenotype of Bim-deficient mice and the post-translational regulation of Bim's pro-apoptotic activity.

3T3 Cells↗
PubMed2000

Direct binding of the Na--H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation.

The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.

Amino Acid Sequence↗
PubMed2000

Transgenic overexpression of human Bcl-2 in islet beta cells inhibits apoptosis but does not prevent autoimmune destruction.

Insulin-dependent diabetes mellitus results when > 90% of the insulin-producing beta cells in the pancreatic islets are killed as a result of autoimmune attack by T cells. During the progression to diabetes, islet beta cells die as a result of different insults from the immune system. Agents such as perforin and granzymes, CD95 ligand and tumor necrosis factor-alpha, or cytokines and free-radicals have all been shown to cause beta cell apoptosis. The anti-apoptotic protein, Bcl-2, might protect against some of these stimuli. We have therefore generated transgenic mice expressing human Bcl-2 in their islet beta cells. Although Bcl-2 was able to prevent apoptosis induced by cytotoxic agents against beta cells in vitro, Bcl-2 alone could not prevent or ameliorate cytotoxic or autoimmune beta cell damage in vivo.

Age of Onset↗
PubMed2000

The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control.

Apoptosis is an evolutionarily conserved process for killing unwanted cells. Genetic and biochemical experiments have indicated that three groups of proteins are necessary for activation of the cell-death effector machinery: cysteine proteases, their adaptors, and proapoptotic Bcl-2 family members. Antiapoptotic Bcl-2 family members are needed for cell survival. We have cloned Bim, a proapoptotic Bcl-2 family member that shares with the family only a 9-16 aa region of homology [Bcl-3 homology region(BH3)], but is otherwise unique. Bim requires its BH3 region for binding to Bcl-2 and activation of apoptosis. Analysis of Bim-deficient mice has shown that Bim is essential for the execution of some but not all apoptotic stimuli that can be antagonized by Bcl-2. Bim-deficient mice have increased numbers of lymphocytes, plasma cells, and myeloid cells, and most develop fatal autoimmune glomerulonephritis. In healthy cells, Bim is bound to the microtubule-associated dynein motor complex, and is thereby sequestered from Bcl-2. Certain apoptotic signals unleash Bim and allow it to translocate to intracellular membranes, where it interacts with Bcl-2 or its homologues. These results indicate that BH3-only proteins are essential inducers of apoptosis that can be unleashed by certain death signals. Unleashed BH3-only proteins neutralize the prosurvival function of Bcl-2-like molecules, and this is thought to liberate Apaf-l-like adapters to activate caspase zymogens, which then initiate cell degradation.

Animals↗
PubMed2000

The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death.

Apoptosis, an evolutionarily conserved process for killing unwanted cells in multicellular organisms, is essential for normal development, tissue homeostasis and as a defense against pathogens. The control of apoptosis is of considerable importance for clinical medicine, as its deregulation can lead to cancer, autoimmunity or degenerative diseases. We have disrupted the Bim gene in the mouse and demonstrated that it plays a major and non-redundant role in embryogenesis, in the control of hematopoietic cell death, and as a barrier against autoimmunity.

Animals↗
PubMed2000

The relationship between elution time and eluate volume using the Ultra-TechneKow DTE technetium-99m generator.

OBJECTIVE: The new Ultra-TechneKow Dry Ship Top Elute 99mTc generator (UTK-DTE generator; Mallinckrodt Medical, Inc., St. Louis, MO) was devised to facilitate fractionated elution with an ergonomically designed elution shield. Fractionation is accomplished traditionally by visually observing the eluted volume through 2 layers of leaded glass windows located in a lighted elution shield and generator auxiliary shield. The goal of our study was to use elution time to determine the endpoint for obtaining the required volume of 99mTc-eluate from a UTK-DTE generator. METHODS: After triplicate elution at several predetermined elution times, the initial weight of the evacuated collecting vial was subtracted from the total weight after elution to determine the elution volume. RESULTS: A quadratic relationship was established between the eluate volume (v, mL) and elution time (t, s) (v = 0.3594 + 0.1889 t - 0.0009 t2). This equation is suitable for use with the 10-mL elution vial. This formula may not be accurate for the first elution since the UTK-DTE generator is a dry-column generator when shipped. The following elution times were calculated for some commonly eluted volumes: 2 mL (9 s), 4 mL (22 s), 5 mL (28 s), 7 mL (45 s), and 10 mL (88 s). CONCLUSION: Our calculated elution time method can be used to predict the eluate volume from a UTK-DTE generator.

Algorithms↗
PubMed1999

Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x(L).

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-x(L) did not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.

Adaptor Proteins, Signal Transducing↗