Sequence requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP. Hid binds Op-IAP in a manner similar to Smac binding of XIAP.

It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. However, there is little direct experimental evidence in support of this hypothesis; indeed, evidence exists from previous studies suggesting that the mode of binding is not similar. We have now precisely mapped the interaction between Hid and Op-IAP, and we show clearly for the first time that the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, our data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid, which may explain some of the earlier contradictory results. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP.     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.     

Cleavage of the apoptosis inhibitor DIAP1 by the apical caspase DRONC in both normal and apoptotic Drosophila cells

In Drosophila S2 cells, the apical caspase DRONC undergoes a low level of spontaneous autoprocessing. Unintended apoptosis is prevented by the inhibitor of apoptosis DIAP1, which targets the processed form of DRONC for degradation through its E3 ubiquitin protein ligase activity. Recent reports have demonstrated that shortly after the initiation of apoptosis in S2 cells, DIAP1 is cleaved following aspartate residue Asp-20 by the effector caspase DrICE. Here we report a novel caspase-mediated cleavage of DIAP1 in S2 cells. In both living and dying S2 cells, DIAP1 is cleaved by DRONC after glutamate residue Glu-205, located between the first and second BIR domains. The mutation of Glu-205 prevented the interaction of DIAP1 and processed DRONC but had no effect on the interaction with full-length DRONC. The mutation of Glu-205 also had a negative effect on the ability of overexpressed DIAP1 to prevent apoptosis stimulated by the proapoptotic protein Reaper or by UV light. These results expand our knowledge of the events that occur in the Drosophila apoptosome prior to and after receiving an apoptotic signal.     

How the Electronic Health Record Will Change the Future of Health Care

Genetic testing is expected to play a critical role in patient care in the near future. Advances in genomic research have the potential to impact medicine in very tangible and direct ways, from carrier screening to disease diagnosis and prognosis to targeted treatments and personalized medicine. However, numerous barriers to widespread adoption of genetic testing continue to exist, and health information technology will be a critical means of addressing these challenges. Electronic health records (EHRs) are a digital replacement for the traditional paper-based patient chart designed to improve the quality of patient care. EHRs have become increasingly essential to managing the wealth of existing clinical information that now includes genetic information extracted from the patient genome. The EHR is capable of changing health care in the future by transforming the way physicians use genomic information in the practice of medicine.     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

Effects of manipulating apoptosis on Sindbis virus infection of Aedes aegypti mosquitoes

Improved control of vector-borne diseases requires an understanding of the molecular factors that determine vector competence. Apoptosis has been shown to play a role in defense against viruses in insects and mammals. Although some observations suggest a correlation between apoptosis and resistance to arboviruses in mosquitoes, there is no direct evidence tying apoptosis to arbovirus vector competence. To determine whether apoptosis can influence arbovirus replication in mosquitoes, we manipulated apoptosis in Aedes aegypti mosquitoes by silencing the expression of genes that either positively or negatively regulate apoptosis. Silencing of the A. aegypti anti-apoptotic gene iap1 (Aeiap1) caused apoptosis in midgut epithelium, alterations in midgut morphology, and 60 to 70% mosquito mortality. Mortality induced by Aeiap1 silencing was rescued by cosilencing the initiator caspase gene Aedronc, indicating that the mortality was due to apoptosis. When mosquitoes which had been injected with Aeiap1 double-stranded RNA (dsRNA) were orally infected with Sindbis virus (SINV), increased midgut infection and virus dissemination to other organs were observed. This increase in virus infection may have been due to the effects of widespread apoptosis on infection barriers or innate immunity. In contrast, silencing the expression of Aedronc, which would be expected to inhibit apoptosis, reduced SINV midgut infection and virus dissemination. Thus, our data suggest that some level of caspase activity and/or apoptosis may be necessary for efficient virus replication and dissemination in mosquitoes. This is the first study to directly test the roles of apoptosis and caspases in determining mosquito vector competence for arboviruses.     

Sindbis Virus Induces Apoptosis through a Caspase-Dependent,CrmA-Sensitive Pathway

Sindbis virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis virus activates the cell suicide program is not known, we demonstrate here that Sindbis virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis virus-induced cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis virus-induced apoptosis.Sindbis virus is an alphavirus of the Togaviridae family which causes encephalitis in mice and thus serves as a model for closely related human encephalitic viruses. Infection of a variety of cultured cell types with Sindbis virus triggers programmed cell death (33). The induction of apoptosis in neurons of mouse brains and spinal cords correlates with the neurovirulence of the virus strain and with mortality in mice, suggesting that induction of apoptosis may be a primary cause of death of young mice (34). In support of this hypothesis, overexpressed inhibitors of apoptosis, such as Bcl-2 and IAP, can protect cultured cells from Sindbis virus-induced apoptosis, and Bcl-2 efficiently reduces mortality in mice (17, 31, 32). These findings also raise the possibility that endogenous inhibitors of apoptosis inhibit Sindbis virus-induced cell death, leading to a persistent virus infection (33, 61). Encephalitis and/or a fatal stress response may be a consequence of neuronal apoptosis (21, 59). Alternatively, there may be multiple pathways that work independently to cause fatal disease.A crucial role for the caspase family of cysteine proteases in the execution phase of programmed cell death is supported by genetic (24, 52, 66), biochemical (29, 57), and physiological (25) evidence. A current model proposes a cascade of events by which caspases proteolytically activate other caspases (35, 39, 46). More recent evidence suggests that different death stimuli trigger the activation of a subset of upstream caspases that possess long prodomains at their N termini (3, 41, 62). These prodomains serve to target proteases to specific protein complexes, where the prodomains are removed by proteolysis to produce active proteases. These caspases proteolytically activate other downstream caspases (with shorter prodomains) that cleave key substrates to ultimately produce the characteristic apoptotic phenotype of cell shrinkage, membrane blebbing, chromatin condensation, oligonucleosomal DNA fragmentation, and cell death (42, 53). A growing list of proteolytic substrates of the caspases have been identified, including protein kinase C delta (18), the retinoblastoma tumor suppressor (56), fodrin (12, 38), lamins (30, 47), the nuclear immunophilin FKBP46 (1), Bcl-2 (7), and several autoantigens (5), and they all are cleaved after an aspartate residue (P1 position). The precise role of these cleavage events is not known, but they may either inactivate key cellular functions or produce cleavage products with pro-death activity. The cleavage product of Bcl-2 is potently proapoptotic (7), and cleavage of a novel protein designated DFF was recently shown to trigger DNA fragmentation during apoptosis (36). These proteolytic events also serve as biochemical markers of apoptosis. Furthermore, cell death can be inhibited with pseudosubstrate inhibitors of the caspases, such as the cowpox virus serpin CrmA (19, 48), and synthetic peptides such as zVAD-FMK (67). The key feature of these inhibitors is an aspartate at the P1 position, consistent with their specificity for caspases.A role for caspases in viral infections is suggested by the finding that baculovirus infection activates an apoptotic cysteine protease in insect cells that is inhibited by the virus-encoded caspase inhibitor p35 (2). Similar work with mutant adenoviruses has suggested that the adenovirus protein E1A activates caspase 3 (CPP32), generating cleaved products of poly(ADP-ribose) polymerase (PARP) (4). In addition, PARP cleavage is detected during infection of mouse neuroblastoma cells with Sindbis virus (60). To further study the role of these proteases in Sindbis virus-induced programmed cell death, we confirmed that Sindbis virus activates cellular caspases and demonstrated the participation of a subset of caspases in the execution of the apoptotic process.