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Brian R Gastman

Publications and source records attributed to Brian R Gastman.

8 recordsLinked to original sources
PubMed2006

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Apoptosis↗
PubMed2005

Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis.

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products. GrB cleaves Mcl-1 after aspartic acid residues 117, 127, and 157, generating C-terminal fragments that all contain BH-1, BH-2, BH-3, and transmembrane domains. These fragments accumulate at an early apoptotic phase but are eliminated by further degradation during the apoptotic process. The major Mcl-1 C-terminal fragment generated by GrB (residues 118-350) was unable to induce or enhance apoptosis when transfected into tumor cells. Instead, this Mcl-1 C-terminal fragment maintained a partial protective capability against GrB-mediated apoptosis via its lower affinity to Bim. In comparison with ectopically expressed full-length Mcl-1, the stably transfected C-terminal fragments of Mcl-1 were less efficiently localized to the mitochondria. Knockdown of Mcl-1, as achieved by transfection with Mcl-1-specific short interfering RNA, resulted in a significant level of apoptosis in the absence of external apoptotic stimulation and, in addition, enhanced the susceptibility of breast carcinoma cells to GrB cytotoxicity. The significance of Bim in this GrB apoptotic cascade was indicated by the marked protection against GrB-mediated apoptosis endowed on these cells through Bim knockdown. Our studies suggest that the disruption of the Mcl-1.Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.

Apoptosis↗
PubMed2005

Caspase inhibition enhances ischemic tolerance of fasciocutaneous flaps.

OBJECTIVES/HYPOTHESIS: To demonstrate the significance of apoptosis in ischemia-reperfusion injury in revascularized fasciocutaneous flaps and test the hypothesis that pharmacologic inhibition of caspases prolongs the allowable primary ischemia time of these flaps. STUDY DESIGN: Animal study using the epigastric flap in adult male Sprague-Dawley rats. METHODS: Fifty-nine rats were treated with the caspase inhibitor (Q-VD-OPH) reconstituted in dimethylsulfoxide (DMSO) (n = 20, 8 mg/kg:0.8 mL/kg), DMSO alone (n = 19, 0.8 mL/kg), or saline (n = 20, 0.8 mL/kg). Treatment was given as a single intraperitoneal injection 30 minutes before starting primary ischemia. Epigastric flaps were subjected to increasing ischemia times followed by reperfusion. The flaps were harvested and analyzed 7 days later, and viability was assessed. Probit statistical analysis was used to determine the critical ischemia time. This was defined as the time point when 50% of the flaps in each group were expected to survive. RESULTS: The calculated critical ischemia times were 8.92 hours (95% confidence interval 7.19-10.47 h) for the saline group, 16.35 hours (95% confidence interval 11.82-19.89 h) for the DMSO group, and 21.73 hours (95% confidence interval 19.39-25.37 h) for the DMSO with Q-VD-OPH group. These differences were significantly different from each other. CONCLUSIONS: Pretreatment of fasciocutaneous flaps with a free radical scavenger alone or in combination with a caspase inhibitor significantly increases the flap's tolerance of primary ischemia. The added benefit of the caspase inhibitor suggests that apoptosis plays an important role in ischemia-reperfusion injury in soft tissue flaps.

Animals↗
PubMed2004

Degradation of Mcl-1 by granzyme B: implications for Bim-mediated mitochondrial apoptotic events.

Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L.

Adenoviridae↗
PubMed2003

Apoptosis and plastic surgery.

Apoptosis, or programmed cell death, is a phenomenon that is integral to development and cellular homeostasis. In the last decade, many of the essential molecules and pathways that control this phenomenon have been elucidated. Because apoptosis is involved in almost all physiologic and pathologic processes, the understanding of its regulation has significant clinical ramifications. This article reviews the basic understanding of programmed cell death in terms of the effector molecules and pathways. Areas of interest to plastic surgeons are reviewed as they pertain to apoptosis. These areas include allotransplantation, craniofacial and limb development, flap survival, wound healing, stem cell science, and physiologic aging. These topics have not yet been studied extensively in the context of cell death. In this review article, other related and more comprehensively studied scientific areas are used to extrapolate their relevance to apoptosis. Apoptosis is an increasingly better understood process. With the knowledge of how programmed cell death is controlled, combined with the improved ability to effectively perform genetic manipulation and to design specific chemical approaches, apoptosis is gaining clinical relevance. In the next few years, practical clinical breakthroughs will help the medical community to understand the phenomenon of apoptosis and how it relates to the needs of patients.

Aging↗
PubMed2002

Granzyme B-mediated degradation of T-cell receptor zeta chain.

We recently reported that the T-cell receptor (TCR)-zeta chain is cleaved by caspase-3 and -7 in apoptotic T lymphocytes or in a cell-free system. We report here that the zeta chain is also a direct substrate for granzyme B (GrB) proteolytic activity. Loss in expression of TCR-zeta was observed in Jurkat T leukemic cells treated by a combination of GrB and a replication-deficient adenovirus. Although the apoptosis initiated in these cells by GrB was significantly reduced by the pancaspase inhibitor Z-VAD-FMK, TCR-zeta degradation was not prevented. These findings suggest that the GrB-mediated degradation of TCR-zeta chain can proceed despite the efficient inhibition of caspase activity. An in vitro translated TCR-zeta product was efficiently cleaved by GrB, which suggests that the TCR-zeta protein is a direct substrate for GrB. As assessed by site-directed mutagenesis, the activity of GrB was directed toward aspartic acid residues that were different from those of recombinant caspase-3. Whereas caspase-3 cleavage products appear to accumulate, the GrB-generated products seem to undergo further degradation, which suggests the presence of multiple GrB-specific cleavage sites within the TCR-zeta protein. These findings suggest that the TCR-zeta protein in target T lymphocytes serves as a substrate for the proteolytic activities that are featured by the two major mechanisms of cytotoxicity: death receptor pathways mediated by caspases and granule exocytosis mediated by direct GrB activity or GrB-activated caspases. TCR-zeta protein degradation may be of significance in cytotoxic mechanisms directed against T cells infected with viruses, such as HIV-1, in which the TCR-zeta protein is used for viral pathogenesis.

Amino Acid Chloromethyl Ketones↗