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M MacFarlane

Publications and source records attributed to M MacFarlane.

At least 37 records · Page 2Linked to original sources
PubMed1999

Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis.

Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochrome c is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells.

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Low levels of p53 are associated with resistance to tetrachlorodibenzo-p-dioxin toxicity in DBA/2 mice.

We show here that DBA/2 strain mice have a complex mutation/polymorphism in the promoter region of the Trp53 locus (the mouse p53 locus). This region has previously been shown to be essential for p53 expression. We further show that the DBA/2 mutation is associated with approximately fourfold lower p53 levels in thymocytes treated with the DNA-damaging agent etoposide in-vitro, and with relative resistance of these thymocytes to apoptosis induced by the DNA-damaging agent etoposide compared with C57BL/6 mice. When part of the promoter containing this mutation was inserted into a plasmid containing a luciferase reporter gene but lacking eukaryote promoter sequences and transfected into MCF-7 human breast cell line cells, the mean luciferase activity was slightly less with the DBA/2 than with the C57BL/6 promoter-reporter construct (p < 0.01). We found that DBA/2xC57BL/6 F2 hybrid mice with the DBA/2 genotype at the Trp53 locus were relatively resistant to tetrachlorodibenzo-p-dioxin toxicity, and this resistance was additive with resistance associated with the Ahr locus. DBA/2 mice are long-lived and do not have particularly high levels of cancer, suggesting either that they carry other compensatory tumour resistance alleles (such as Ahr(d)), or that, while there may be a p53 protein dosage effect for acute toxicity, lower than normal levels of p53 may still be sufficient to protect against cancer. In evolutionary terms, it may be better to maintain low levels of p53 in order to avoid death from acute toxicity, even at the expense of a higher incidence of cancer in later life.

Animals↗
PubMed1999

Surgeon-tool force/torque signatures--evaluation of surgical skills in minimally invasive surgery.

The best method of training for laparoscopic surgical skills is controversial. Some advocate observation in the operating room, while others promote animal and simulated models or a combination of surgical related tasks. The mode of proficiency evaluation common to all of these methods has been subjective evaluation by a skilled surgeon. In order to define an objective means of evaluating performance, an instrumented laparoscopic grasper was developed measuring the force/torque at the surgeon hand/tool interface. The measured database demonstrated substantial differences between experienced and novice surgeon groups. Analyzing forces and torques combined with the state transition during surgical procedures allows an objective measurement of skill in MIS. Teaching the novice surgeon to limit excessive loads and improve movement efficiency during surgical procedures can potentially result in less injury to soft tissues and less wasted time during laparoscopic surgery. Moreover the force/torque database measured in this study may be used for developing realistic virtual reality simulators and optimization of medical robots performance.

Algorithms↗
PubMed1998

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver.

Caspases plays a key role in the execution phase of apoptosis. "Initiator" caspases, such as caspase-8, activate "effector" caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits.

Caspases play a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. Ectopic overexpression of the short prodomain caspases-3 and -6 precursors in mammalian cells does not induce apoptosis. This is due to their inability to undergo autocatalytic processing/activation and suggests that they depend on the long prodomain caspases for activation. To investigate directly the apoptotic activity of these two caspases in vivo, we engineered constitutively active recombinant caspases-3 and -6 precursors. This was achieved by making contiguous precursor caspases-3 and -6 molecules, which have their small subunits preceding their large subunits. Unlike their wild type counterparts, these recombinant molecules were capable of autocatalytic processing in an in vitro translation reaction, suggesting that they are catalytically active. They were also capable of autoprocessing and inducing apoptosis in vivo independent of the upstream caspases. Furthermore, their autocatalytic and apoptotic activities were inhibited by the pancaspase inhibitor z-VAD-fluoromethylketone, but not by CrmA or Bcl-2, thus directly demonstrating that the targets of inhibition of apoptosis by CrmA and Bcl-2 are upstream of caspases-3 and -6. Since caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases could be used at very low concentrations in gene therapy model systems to induce apoptosis in target tissues or tumors.

Amino Acid Chloromethyl Ketones↗
PubMed1998

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases.

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Computerized endoscopic surgical grasper.

We report a computerized endoscopic surgical grasper with computer control and a force feedback (haptic) user interface. The system uses standard unmodified grasper shafts and tips. The device can control grasping forces either by direct surgeon control, via teleoperation, or under software control. In this paper, we test an automated palpation function in which the grasper measures mechanical properties of the grasped tissue by applying a programmed series of squeezes. Experimental results show the ability to discriminate between the normal tissues of small bowel, lung, spleen, liver, colon, and stomach. We anticipate applications in telesurgery, clinical endoscopic surgery, surgical training, and research.

Endoscopes↗
PubMed1997

Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL.

A human receptor for the cytotoxic ligand TRAIL (TRAIL receptor-1, designated DR4) was identified recently as a member of the tumor necrosis factor receptor family. In this report we describe the identification of two additional human TRAIL receptors, TRAIL receptor-2 and TRAIL receptor-3, that belong to the tumor necrosis factor receptor family. Interestingly, TRAIL receptor-2 but not TRAIL receptor-3 contains a cytoplasmic "death domain" necessary for induction of apoptosis and is hence designated death receptor-5 (DR5). Like DR4, DR5 engages the apoptotic pathway independent of the adaptor molecule FADD/MORT1. Because of its lack of a death domain, TRAIL receptor-3 is not capable of inducing apoptosis. However, by competing for TRAIL, it is capable of inhibiting TRAIL-induced apoptosis. Thus, TRAIL receptor-3 may function as an antagonistic decoy receptor to attenuate the cytotoxic effect of TRAIL in most tissues that are TRAIL+, DR4+, and DR5+.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells.

Identification of the processing/activation of multiple interleukin-1beta converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3alpha but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3alpha. was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3alpha or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3alpha, Mch2alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.

Apoptosis↗
PubMed1997

Activation of CPP32 and Mch3 alpha in wild-type p53-induced apoptosis.

DNA-damaging agents induce apoptosis primarily by a p53-dependent pathway. LTR6 cells containing a temperature-sensitive p53 were used to dissect further the mechanisms of p53-induced apoptosis. Apoptosis was accompanied by the processing and activation of CPP32 and Mch3 alpha, together with the cleavage of poly(ADP-ribose) polymerase and lamin B1. These results demonstrate a critical role for the activation of interleukin-1 beta-converting enzyme-like proteases in p53-induced apoptosis.

Animals↗
PubMed1996

Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

Amino Acid Chloromethyl Ketones↗
PubMed1996

DNA-damaging agents induce both p53-dependent and p53-independent apoptosis in immature thymocytes.

Apoptosis in the immature thymus can be induced by both p53-dependent and -independent pathways, the former being activated by exposure to DNA-damaging agents and the latter being induced by glucocorticoids [Nature (Lond.) 362:847-849; Nature (Lond.) 362:849-852 (1993)]. We report that the DNA-damaging agents etoposide and gamma-radiation induced similar levels of apoptosis in both proliferatively enriched and quiescent immature rat thymocytes, as assessed by flow cytometry and the formation of both kilobase-pair and 180-bp integer fragments of DNA. However, a marked stabilization of p53 occurred exclusively in the proliferatively enriched population, which was also enriched for immature CD4- CD8- and mature CD4+ CD8-/CD4- CD8+ cells. In contrast, DNA damage-induced apoptosis in quiescent mature peripheral T cells was associated with an accumulation of p53. Our studies suggest that stabilization of p53 in thymocytes in response to DNA damage may be developmentally regulated. In immature thymocytes obtained from p53-null mice, DNA-damaging agents induced apoptosis at significantly lower levels and at later times than that seen in cells from p53 wild-type animals. These data support the hypothesis that DNA-damaging agents induce apoptosis primarily via a p53-dependent pathway in immature thymocytes as previously reported. We report here that DNA damage can also induce apoptosis by a p53-independent pathway in a particular subpopulation of immature thymocytes.

Amino Acid Sequence↗
PubMed1995

DNA degradation and proteolysis in thymocyte apoptosis.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD.FMK) inhibit apoptosis. N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) has a very different effect, inducing the early morphological and biochemical changes associated with apoptosis. TLCK inhibits trypsin-like proteases whilst Z-VAD.FMK inhibits interleukin-1 beta-converting enzyme (ICE)-like proteases; this and the contrasting effects of TPCK support the hypothesis that thymocyte apoptosis involves a hierarchy of proteases which act at different stages of the process.

Animals↗
PubMed1994

Cdc2 activation is not required for thymocyte apoptosis.

Apoptosis is a mode of cell death characterized by chromatin condensation and disassembly of the nuclear lamina, processes that are also characteristic of mitosis. The apparent similarity between the two events, together with observations that apoptosis can occur following G2 arrest, has led to the suggestion that apoptosis could be a defective form of mitosis. Further support for this idea comes from the recent description of activation of the p34cdc2 protein kinase (Cdc2), the universal M-phase promoter, during death induced by a lymphocyte granule protease. We have monitored the protein kinase activity of Cdc2 during apoptosis of primary rat thymocytes, which die from a quiescent (G0) state. We demonstrate unequivocally that activation of Cdc2 is not involved in the induction of apoptosis in thymocytes, indicating that chromatin condensation and lamina disassembly occur in this system by processes different from those that operate in mitosis.

Animals↗
PubMed1994

Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes.

Apoptosis is a process in which cells die in a controlled manner and apparently participate in their own demise. It is best characterized morphologically by condensation of chromatin and biochemically by cleavage of chromatin at internucleosomal regions to yield a classical DNA ladder pattern. Apoptosis was induced in thymocytes by exposure to either the glucocorticoid, dexamethasone, or DNA topoisomerase II inhibitor, etoposide. We describe the formation of large m.w. fragments of DNA, 30 to 50 kilobase pairs in length, in a population of these thymocytes at an early stage of apoptosis before internucleosomal cleavage of DNA. These fragments are absent in normal thymocytes and their formation is dependent on protein synthesis. Their appearance is coincident with the commitment of these cells to apoptosis. The formation of these large fragments is associated with the condensation of chromatin, abutting the nuclear membrane, recognized as one of the earliest ultrastructural signs of apoptosis. Subsequent cleavage of these large fragments gives rise to oligonucleosomal fragments and is independent of protein synthesis. We propose that the formation of large fragments of DNA represents a key committed step in apoptosis, and that it is from these fragments that the archetypal DNA ladders associated with apoptosis are derived.

Animals↗