Quantitative chemoproteomic profiling reveals multiple target interactions of spongiolactone derivatives in leukemia cells.

The spongiolactones are marine natural products with an unusual rearranged spongiane skeleton and a fused ß-lactone ring. These compounds have potential anticancer properties but their mode of action has yet to be explored. Here we employ activity-based protein profiling to identify the targets of a more potent spongiolactone derivative in live cancer cells, and compare these to the targets of a simpler ß-lactone. These hits provide the first insights into the covalent mechanism of action of this natural product class.

Does body mass index influence the degree of pelvic tilt produced by a Crawford wedge?

BACKGROUND: A pelvic tilt of 15° is standard practice when positioning a woman for caesarean section, and is commonly produced by tilting the operating table or placing a wedge under the right hip. This study investigated whether body mass index affects the degree of pelvic tilt produced when a wedge is used. METHODS: Women undergoing category 3 and 4 caesarean sections were stratified into three groups according to their body mass index at antenatal booking: ≤ 25kg/m(2), 25.1-35kg/m(2) and >35kg/m(2). Twenty women were recruited into each group. Lateral tilt at caesarean section was provided with a Crawford wedge under the right hip and the degree of pelvic tilt was measured using a protractor device. RESULTS: The median [range] pelvic tilt angle for the groups in order of ascending body mass index were 15° [12-22°], 19° [11-29°] and 17° [2-28°]. There was a significant increase in the variability of pelvic tilt with increasing body mass index (P=0.001). The proportion of patients with pelvic tilt <15° was observed to be 20%, 15% and 30% for women of body mass index ≤ 25kg/m(2), 25.1-35kg/m(2) and >35kg/m(2), respectively. CONCLUSION: Variability in pelvic tilt increased with body mass index and was greatest with a booking body mass index >35kg/m(2).

A comparison of the pelvic angle applied using lateral table tilt or a pelvic wedge at elective caesarean section.

Lateral table tilt or a pelvic wedge are commonly used to reduce inferior vena cava compression during obstetric anaesthesia in the supine position. Direct measurement of pelvic angle allows individual assessment of the effectiveness of these manoeuvres in achieving a tilted position. We observed routine practice during caesarean section after random allocation to one or other of these methods. The anaesthetist managing the case was asked to position the women after induction of spinal anaesthesia using either left table tilt or a wedge under the right hip. We then measured pelvic angle in all women, and the table angle in women who had table tilt. The mean (SD [range]) pelvic angle was 20.2° (8.1° [9°-37°]) in 18 women with table tilt and 21.0° (7.5° [10°-36°]) in 17 women with a wedge. The mean (SD [range]) table angle was 12.4° (3.1° [8°-21°]) in the women with table tilt. There was a significant difference between table angle and pelvic angle in the women with table tilt (p = 0.0003), but no significant difference in pelvic angle between the table tilt and wedge groups. Measurement of table angle does not represent pelvic position adequately in the majority of women. However, this study showed that lateral table tilt and a pelvic wedge were equally effective in producing tilt of the pelvis.

Transmission of two Australian strains of murine cytomegalovirus (MCMV) in enclosure populations of house mice (Mus domesticus).

To control plagues of free-living mice (Mus domesticus) in Australia, a recombinant murine cytomegalovirus (MCMV) expressing fertility proteins is being developed as an immunocontraceptive agent. Real-time quantitative PCR was used to monitor the transmission of two genetically variable field strains of MCMV through mouse populations after 25% of founding mice were infected with the N1 strain, followed by the G4 strain 6 weeks later. Pathogen-free wild-derived mice were released into outdoor enclosures located in northwestern Victoria (Australia). Of those mice not originally inoculated with virus, N1 DNA was detected in more than 80% of founder mice and a third of their offspring and similarly, G4 DNA was detected in 13% of founder mice and in 3% of their offspring. Thus, prior immunity to N1 did not prevent transmission of G4. This result is promising for successful transmission of an immunocontraceptive vaccine through Australian mouse populations where MCMV infection is endemic.

Species-specificity of a murine immunocontraceptive utilising murine cytomegalovirus as a gene delivery vector.

Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) expressing the mouse egg-coat protein zona pellucida 3 (mZP3) has been shown to sterilise female mice by breaking self-tolerance and inducing an immune response against the host ZP3. This virus has the potential to be used for mouse population control, however the effect of this recombinant immunocontraceptive virus in non-host species must be determined. Recombinant MCMV-mZP3, based on both laboratory and wild strains of virus, induced long-lived antibody responses against structural viral proteins and mZP3 when inoculated into laboratory rats, although no viral DNA or replicating virus was identified. The anti-mZP3 antibodies were specific for mouse ZP3, did not cross-react with rat ZP3, and had no effect on the fertility of the rats.

Characterization of the Drosophila caspase, DAMM.

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced in Escherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels of damm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.

Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD.

RAIDD, a caspase recruitment domain (CARD) containing molecule, interacts with procaspase-2 in a CARD-dependent manner. This interaction has been suggested to mediate the recruitment of caspase-2 to the tumour necrosis factor receptor 1 (TNFR1). In this paper we have studied the subcellular localization of RAIDD and its interaction with caspase-2. We demonstrate that endogenous RAIDD is mostly localized in the cytoplasm and to some extent in the nucleus. RAIDD localization is not affected by TNF-treatment of HeLa cells, but in cells ectopically expressing caspase-2, a fraction of RAIDD is recruited to the nucleus. In transfected cells, coexpression of RAIDD and caspase-2 leads to CARD-dependent colocalization of the two proteins to discrete subcellular structures. We further show that overexpression of the RAIDD-CARD results in the formation of filamentous structures due to CARD-mediated oligomerization. These structures were similar to death effector filaments (DEFs) formed by FADD and FLICE death effector domains (DEDs), and partially colocalized with DEFs. Our results suggest that similar to the DED, the RAIDD-CARD has the ability to form higher order complexes, believed to be important in apoptotic execution. We also present evidence that RAIDD-CARD oligomerization may be regulated by intramolecular folding of the RAIDD molecule.

The role of caspases in apoptosis.

The process of apoptosis, or programmed cell death, is fundamental during normal development and homeostasis and aberrant apoptosis has been implicated in a number of human diseases. The cellular machinery involved in the execution of apoptosis includes a family of cysteine proteases termed caspases. Caspases exhibit the rare substrate preference of cleavage C-terminal to aspartate residues, a property shared only by the cytotoxic lymphocyte serine protease, granzyme B. Experimental evidence demonstrates a vital role for caspase activation in the apoptotic pathway, and, as such, caspases are a target for the development of agents that can modulate their activity. This article reviews the members of the caspase family and the role that each contributes to the execution of cell death induced by apoptotic stimuli.

Conversion of procaspase-3 to an autoactivating caspase by fusion to the caspase-2 prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases.

Prodomain-dependent nuclear localization of the caspase-2 (Nedd2) precursor. A novel function for a caspase prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis by cleaving several key cellular proteins. Despite their function in apoptosis, little is known about where in the cell they are localized and whether they are translocated to specific cellular compartments upon activation. In the present paper, using Aequorea victoria green fluorescent protein fusion constructs, we have determined the localization of Nedd2 (mouse caspase-2) and show that both precursor and processed caspase-2 localize to the cytoplasmic and the nuclear compartments. We demonstrate that the nuclear localization of caspase-2 is strictly dependent on the presence of the prodomain. A caspase-2 prodomain-green fluorescent protein localized to dot- and fiber-like structures mostly in the nucleus, whereas a protein lacking the prodomain was largely concentrated in the cytoplasm. We also show that an amino-terminal fusion of the prodomain of caspase-2 to caspase-3 mediates nuclear transport of caspase-3, which is normally localized in the cytoplasm. These results suggest that, in addition to roles in dimerization and recruitment through adaptors, the caspase-2 prodomain has a novel function in nuclear transport.

Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis.

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma-radiation, tumor necrosis factor-alpha, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237 downward arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl-terminal regions.

Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway. The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli. In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo. We demonstrate that the expression of pro-Nedd2 in yeast cells results in processing of the precursor. A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues. Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing. In vitro processing of pro-Nedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur. Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.

Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis.

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.

Processing of the Nedd2 precursor by ICE-like proteases and granzyme B.

BACKGROUND: The Nedd2/Ich-1 protein belongs to a growing family of mammalian cysteine proteases similar to interleukin-1 beta converting enzyme (ICE). Because of their similarity to the Cacnorhabditis elegans cell death protein CED-3, the ICE-like proteins are thought to play a key role in the execution of apoptosis. The active form of ICE is a tetramer consisting of two heterodimers (p20 + p10)2 derived from the cleavage of the pro-enzyme. RESULTS: In the present communication we show that the p51 Nedd2 precursor (pro-Nedd2) is also cleaved into p20-like (p19) and p10-like (p12) subunits by extracts prepared from cultured cell lines. Extracts from apoptotic NIH-3T3 cells but not normal growing NIH-3T3 cells also contained pro-Nedd2 cleaving activity. The processing of pro-Nedd2 by cell extracts was inhibited by characteristic inhibitors of ICE-like proteases. Additionally we show that pro-Nedd2 (p51) can be processed in vitro by active CPP32 and ICE, and to a lesser extent by Mch2 and Nedd2. Granzyme B, a serine protease required for cytotoxic T lymphocyte (CTL) mediated killing of target cells, also cleaved pro-Nedd2 to p19 + p12 subunits. CONCLUSIONS: Our observations suggest that Nedd2 activation requires cleavage by one or more ICE-like proteases that lie upstream in the proteolytic cascade. Cleavage of pro-Nedd2 by granzyme B indicates that Nedd2 may be one of the downstream effectors in the CTL-mediated killing of target cells.

Increased expression of c-Kit or its ligand Steel Factor is not a common feature of adult acute myeloid leukaemia.

Cell surface levels of the receptor tyrosine kinase P145(c-kit), the product of the c-kit proto-oncogens, in a panel of 80 primary adult acute myeloid leukaemia (AML) specimens collected at presentation were quantitated by immunofluorescence and flow cytometry, and compared with levels on CD34+ bone marrow cells from normal donors. Receptor levels on AML blast cells were extremely variable and were similar to, or less than, those on normal stem and progenitor cells. In general P145(c-kit) expression was higher on cells of immature phenotype (FAB M1 and M2). c-kit mRNA was quantitated by ribonuclease protection assay (RPA) and was shown to be correlated with cell surface protein expression (r=0.76; P<0.001). This indicates that ligand-mediated receptor internalisation or other mechanisms of increased protein turnover are not responsible for variations in the level of P145(c-kit) in AML specimens. Quantitative Southern blotting was used to examine c-kit gene copy number in 25 of these specimens and was found to be normal in all but one. Thus we have found little evidence of over-expression of c-kit in adult AML. mRNA for the c-kit ligand, Steel Factor (SLF) was also quantitated by RPA in these specimens. While SLF message was detectable (limit of detection approximately 10(4) copies per 10 microgram total RNA; equivalent to 1 copy per 100 cells) in 19% of cases, these specimens in general contained low levels of c-kit mRNA. Thus, an autocrine cycle involving c-kit and SLF does not appear to be a common feature of AML.

Role of multiple cellular proteases in the execution of programmed cell death.

A family of mammalian homologues of the Caenorhabditis elegans cell death protein Ced-3 has been recently discovered. These mammalian proteins encode novel cysteine proteases with homology to the interleukin-1 beta converting enzyme (ICE). Although several studies support a role for one or more of these proteases in mediating apoptosis, their mechanism of action is far from understood. The presence of multiple mammalian ICE-like proteases, with apparently similar apoptotic function indicates that, despite its conservation during evolution, the cell death pathway is much more complex in mammals than in the worm. In addition to ICE-like proteases, several other proteases of different cleavage specificities have been implicated in apoptosis. There is now a growing body of evidence suggesting that apoptosis involves the activation of a cascade of proteases. This article summarises the presently available evidence and discusses how multiple proteases might be required in the effector phase of cell death.