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J Marie Hardwick

Publications and source records attributed to J Marie Hardwick.

32 records · Page 2Linked to original sources
PubMed2004

Regulation of cell death in the lymphoid system by Bcl-2 family proteins.

Programmed cell death is an ordered process that is essential for the normal development and homeostasis of an organism. Dysregulation of this programmed pathway, resulting in either excess cell numbers or unscheduled cell death, underlies a number of disease states. Bcl-2 family proteins play a key role in regulating cell death and survival, and a number of studies have demonstrated their role as important regulators of cell fate in the lymphoid system. Mice that are genetically deficient or overexpress various Bcl-2 family proteins have provided important clues regarding their roles in lymphocyte development, progression of lymphoid tumors and analogous human disorders. In addition, lymphotropic viruses may trigger cell proliferation and inhibit cell death with the help of their own Bcl-2 homologues. Comparing the shared and distinct functions of viral and cellular Bcl-2-related proteins yields new insight into their fundamental mechanisms.

Animals↗
PubMed2003

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies.

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-x(L), prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-x(L) with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8(+) T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA showed the greatest increase in E7-specific CD8(+) T cells ( approximately 13-fold increase). This combination of strategies resulted in increased CD8(+) T cell functional avidity, an increased E7-specific CD4(+) Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-x(L) DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.

Adjuvants, Immunologic↗
PubMed2003

Modulation of synaptic transmission by the BCL-2 family protein BCL-xL.

BCL-2 family proteins are known to regulate cell death during development by influencing the permeability of mitochondrial membranes. The anti-apoptotic BCL-2 family protein BCL-xL is highly expressed in the adult brain and localizes to mitochondria in the presynaptic terminal of the adult squid stellate ganglion. Application of recombinant BCL-xL through a patch pipette to mitochondria inside the giant presynaptic terminal triggered multiconductance channel activity in mitochondrial membranes. Furthermore, injection of full-length BCL-xL protein into the presynaptic terminal enhanced postsynaptic responses and enhanced the rate of recovery from synaptic depression, whereas a recombinant pro-apoptotic cleavage product of BCL-xL attenuated postsynaptic responses. The effect of BCL-xL on synaptic responses persisted in the presence of a blocker of mitochondrial calcium uptake and was mimicked by injection of ATP into the terminal. These studies indicate that the permeability of outer mitochondrial membranes influences synaptic transmission, and they raise the possibility that modulation of mitochondrial conductance by BCL-2 family proteins affects synaptic stability.

Adenosine Triphosphate↗
PubMed2003

Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper.

Members of the California serogroup of bunyaviruses (family Bunyaviridae) are the leading cause of pediatric viral encephalitis in North America. Significant cell death is observed as part of the infection pathology. We now report that a Bunyaviral nonstructural protein termed NSs shows sequence similarity to Reaper, a proapoptotic protein from Drosophila. Although NSs proteins lack the Reaper N-terminal motif critical for IAP inhibition, they do retain other functions of Reaper that map to conserved C-terminal regions. Like Reaper, NSs proteins induce mitochondrial cytochrome c release and caspase activation in cell-free extracts and promote neuronal apoptosis and mortality in a mouse model. Independent of caspase activation, Bunyavirus NSs proteins also share with Reaper the ability to directly inhibit cellular protein translation. We have found that the shared capacity to inhibit translation and induce apoptosis resides in common sequence motifs present in both Reaper and NSs proteins. Data presented here suggest that NSs induce apoptosis through a mechanism similar to that used by Reaper, as both proteins bind to an apoptotic regulator called Scythe and can relieve Scythe inhibition of Hsp70. Thus, bunyavirus NSs proteins have multiple Reaper-like functions that likely contribute to viral pathogenesis by promoting cell death and/or inhibiting cellular translation.

Amino Acid Motifs↗
PubMed2003

Viral modulators of cell death provide new links to old pathways.

By observing how viruses facilitate their parasitic relationships with host cells, we gain insights into key regulatory pathways of the cell. Not only are mitochondria key players in the regulation of programmed cell death, but many viral regulators of cell death also alter mitochondrial functions either directly or indirectly. Although cytomegalovirus vMIA and Epstein-Barr virus BHRF1 seem to have opposite effects on mitochondrial morphology, they both inhibit cell death. Drosophila Reaper, a regulator of developmental cell death, acts on IAP (inhibitor of apoptosis) proteins to activate caspases, but can regulate mitochondrial permeability in vitro. Despite its pivotal role in Drosophila, homologues of Reaper in other species were not previously known. Recently, amino acid sequence similarity was recognized between Drosophila Reaper and a protein known to be important for the replication and virulence of mosquito-borne bunyaviruses that cause human encephalitis. Thus, viral mechanisms for regulating apoptosis are diverse and not fully elucidated but promise to provide new insights.

Animals↗
PubMed2003

A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures.

The overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems. In this study, we have evaluated the Bcl-2 homologue, Bcl-x(L) and compared its effectiveness to a Bcl-x(L) mutant lacking most of the non-conserved unstructured loop domain, Bcl-x(L)Delta (deletion of amino acids 26 through 83). The cell line, Chinese hamster ovary (CHO), was genetically modified to express constitutively Bcl-x(L) or the Bcl-x(L) variant and subjected to model apoptotic insults including Sindbis virus (SV) infection, gradual serum withdrawal, and serum deprivation. When cells were engineered to overexpress Bcl-x(L)Delta, cell death due to the SV was inhibited, and Bcl-x(L)Delta provided comparable protection to the wild-type Bcl-x(L) even though expression levels were much lower for the mutant. Furthermore, the cells expressing Bcl-x(L)Delta continued to proliferate following infection while CHO-bcl-x(L) ceased proliferation immediately following infection. As a result, total production of a heterologous protein encoded on the SV was highest in cell lines expressing Bcl-x(L)Delta. Cells expressing the variant Bcl-x(L) also continued to proliferate and showed increased viable cell numbers following gradual serum withdrawal. In contrast, wild-type Bcl-x(L) expressing CHO cells were found to arrest growth but maintain viability following serum withdrawal. Interestingly, CHO cells expressing Bcl-x(L)Delta were also able to recover and return to rapid growth rates much faster than either the wild-type CHO-bcl-x(L) or CHO following the replenishment of fresh complete medium containing 10% FBS. Confocal imaging of yellow fluorescent protein (YFP) fused to the N terminus of Bcl-x(L) and Bcl-x(L)Delta indicated dense aggregates of the Bcl-x(L)Delta while the wild-type protein was distributed throughout the cell in a manner resembling transmembrane localization. As an alternative to complete removal of the loop domain, Bcl-x(L) variants were created in which aspartate residues containing potential caspase recognition sites within the loop domain of Bcl-x(L) were removed. Cell populations expressing various Bcl-x(L)-Asp mutants were exposed to an apoptotic spent medium stimulus, and the cells expressing these Bcl-x(L) variants provided increased viabilities as compared to cells containing wild-type Bcl-x(L) protein. These studies indicate that modification of anti-apoptotic genes can affect multiple cellular properties including response to apoptotic stimuli and cell growth. This knowledge can be valuable in the design of improved apoptosis inhibitors for biotechnology applications.

Alphavirus Infections↗
PubMed2003

BAK alters neuronal excitability and can switch from anti- to pro-death function during postnatal development.

BAK is a pro-apoptotic BCL-2 family protein that localizes to mitochondria. Here we evaluate the function of BAK in several mouse models of neuronal injury including neuronotropic Sindbis virus infection, Parkinson's disease, ischemia/stroke, and seizure. BAK promotes or inhibits neuronal death depending on the specific death stimulus, neuron subtype, and stage of postnatal development. BAK protects neurons from excitotoxicity and virus infection in the hippocampus. As mice mature, BAK is converted from anti- to pro-death function in virus-infected spinal cord neurons. In addition to regulating cell death, BAK also protects mice from kainate-induced seizures, suggesting a possible role in regulating synaptic activity. BAK can alter neurotransmitter release in a direction consistent with its protective effects on neurons and mice. These findings suggest that BAK inhibits cell death by modifying neuronal excitability.

Age Factors↗
PubMed2003

Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins.

Intradermal vaccination by gene gun efficiently delivers DNA vaccines into DCs of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. DCs, however, have a limited life span, hindering their long-term ability to prime antigen-specific T cells. We reason that a strategy that prolongs the survival of DNA-transduced DCs will enhance priming of antigen-specific T cells and DNA vaccine potency. Here we show that codelivery of DNA encoding inhibitors of apoptosis (BCL-xL, BCL-2, XIAP, dominant negative caspase-9, or dominant negative caspase-8) with DNA encoding model antigens prolongs the survival of transduced DCs. More importantly, vaccinated mice exhibited significant enhancement in antigen-specific CD8+ T cell immune responses, resulting in a potent antitumor effect against antigen-expressing tumors. Among these antiapoptotic factors, BCL-xL demonstrated the greatest enhancement in antigen-specific immune responses and antitumor effects. Thus, coadministration of DNA vaccines with DNA encoding antiapoptotic proteins represents an innovative approach to enhance DNA vaccine potency.

Animals↗
PubMed2002

Bax-type apoptotic proteins porate pure lipid bilayers through a mechanism sensitive to intrinsic monolayer curvature.

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and DeltaN76Bcl-x(L) (the Bax-like cleavage fragment of Bcl-x(L)) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or DeltaN76Bcl-x(L). Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.

Apoptosis↗
PubMed2002

Inhibitor specificity of recombinant and endogenous caspase-9.

Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death.

Amino Acid Chloromethyl Ketones↗
PubMed2002

BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis.

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.

Alphavirus Infections↗
PubMed2002

Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins.

Cellular BCL-2 family proteins can inhibit or induce programmed cell death in part by counteracting the activity of other BCL-2 family members. All sequenced gammaherpesviruses encode a BCL-2 homologue that potently inhibits apoptosis and apparently escapes some of the regulatory mechanisms that govern the functions of their cellular counterparts. Examples of these protective proteins include BHRF1 of Epstein-Barr virus (EBV) and KSBcl-2 of Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8. The gamma-1 subgroup of these viruses, such as EBV, encodes a second BCL-2 homologue. We have now found that this second BCL-2 homologue encoded by EBV, BALF1, inhibits the antiapoptotic activity of EBV BHRF1 and of KSBcl-2 in several transfected cell lines. However, BALF1 failed to inhibit the cellular BCL-2 family member, BCL-x(L). Thus, BALF1 acts as a negative regulator of the survival function of BHRF1, similar to the counterbalance observed between cellular BCL-2 family members. Unlike the cellular BCL-2 family antagonists, BALF1 lacked proapoptotic activity and could not be converted into a proapoptotic factor in a manner similar to cellular BCL-2 proteins by caspase cleavage or truncation of the N terminus. Coimmunoprecipitation experiments and immunofluorescence assays suggest that a minimal amount, if any, of the BHRF1 and BALF1 proteins colocalizes inside cells, suggesting that mechanisms other than direct interaction explain the suppressive function of BALF1.

Amino Acid Sequence↗
PubMedDate not supplied

Actions of BAX on mitochondrial channel activity and on synaptic transmission.

Changes in mitochondrial architecture and permeability facilitate programmed cell death. The BCL-2 family protein BAX is implicated in the formation of large "death channels" in outer mitochondrial membranes. We found that BAX-induced channels on mitochondria may have alternative functions. By patch clamping mitochondrial membranes inside the presynaptic terminal of the living squid giant synapse, we made direct measurements of channel activity produced by BAX application. Only infrequently did BAX application result in large conductance channels similar to those produced by a proapoptotic BCL-xL fragment or by application of a BH3-only peptide. Instead, the majority of outer mitochondrial channels induced by BAX had much smaller conductances than those found previously for the proapoptotic protein. Injection of BAX into the presynaptic terminal did not abolish synaptic transmission, contrary to previous findings with the proapoptotic fragment of BCL-xL. Instead, injection of BAX caused an increase in neurotransmitter release, as has also been found for the full-length antiapoptotic BCL-xL protein. We suggest that BAX can act to enhance synaptic efficacy in a normal physiological setting. Furthermore, the occasional large openings may reflect the function of "activated" BAX either to facilitate cell death or to play a physiological role in decreasing synaptic activity.

Animals↗