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Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

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Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium (www.cebiond.org), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

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The article presents readers' contribution on addictions including drug addiction of Deidre McDonald from Dickinson, Texas, cigarettes addiction of an unamed reader from St. Augustine, Florida, and the addiction of love by Theresa Dyer from Patchogue, New York.

The article features several plant explorers and botanists, including Thomas Drummond, Ferdinand Jacob Lindheimer and Josiah Gregg, and their contributions in botany. It mentions the plants that were discovered by and were named after the botanists including Drummond's phlox, Lindheimer's senna and Gregg's mistflower. It also announces that the Lady Bird Johnson Wildflower Center's biannual plant sale is on April 11 to 13, 2014.

Unicellular eukaryotes have been suggested as undergoing self-inflicted destruction. However, molecular details are sparse compared with the mechanisms of programmed/regulated cell death known for human cells and animal models. Here, we report a molecular cell death pathway in Saccharomyces cerevisiaeleading to vacuole/lysosome membrane permeabilization. Following a transient cell death stimulus, yeast cells die slowly over several hours, consistent with an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 complex, a vesicle trafficking adapter known to transport and install newly synthesized proteins on the vacuole/lysosome membrane. To promote cell death, AP-3 requires its Arf1-GTPase-dependent vesicle trafficking function and the kinase Yck3, which is selectively transported to the vacuole membrane by AP-3. Video microscopy revealed a sequence of events where vacuole permeability precedes the loss of plasma membrane integrity. AP-3-dependent death appears to be conserved in the human pathogenic yeast Cryptococcus neoformans.

Administrator influence produced an increase in correct identifications rates (with little effect on confidence) and increased false identification rates (with a decrease in confidence).

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. Antioxid.Redox Signal. 7, 1092–1100.

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.

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 ΔN76Bcl-xL(the Bax-like cleavage fragment of Bcl-xL) 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 ΔN76Bcl-xL. 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.

ABSTRACTCellular 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.

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.

ABSTRACTCellular 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-xL. 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.

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild-type Bcl-2 was compared to a Bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA “ladder” and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl-2 mutant, cell death due to Sindbis virus was inhibited in a concentration-dependent manner. Furthermore, the Bcl-2 mutant provided increased protection as compared to wild-type Bcl-2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl-2 variants compared to the parental cell line. In order to understand the reasons for the improved anti-apoptosis properties of the mutant, wild-type Bcl-2 and mutant Bcl-2 were examined by Western blot following each model insult. Wild-type Bcl-2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl-2 protein was not degraded during the same period. The processing of Bcl-2 was found to correlate with reduced cell viabilities following the two external insults to suggest that Bcl-2 degradation may limit its ability to inhibit apoptosis. These studies indicate that the cells regulate anti-apoptosis protein levels and these processing events can limit the effectiveness of cell death inhibition strategies in mammalian cell culture systems. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 211–222, 2001.

Lisianthus (Eustoma grandiflorum Grise.) with novel flower colour patterns have been generated by genetic modification techniques that alter flavonoid biosynthesis. As described previously, the level and pattern of pigmentation in purple-flowered lisianthus cultivars was altered by the introduction of a lisianthus chalcone synthase (CHS) cDNA in an antisense orientation. As the next step towards commercial development of these new cultivars, the consistency of altered flower patterns in the progeny and the performance of these lines under conditions resembling those used by commercial lisianthus growers in New Zealand have now been assessed. The introduced antisense CHS transgene had no apparent effect on germination rates or plant survival. Altered flower patterns observed in the progeny correlated with the inheritance of the transgenes, as measured by kanamycin resistance and reduction in CHS expression. At least two of the lines showed sufficient consistency in flower pattern to warrant continued development. Additionally, no transfer of the introduced DNA between transgenic and neighbouring non-transformed lisianthus was observed during this trial.

ABSTRACTThe antiapoptotic Bcl-2 and Bcl-xLproteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966–1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554–559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (?HV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by ?HV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-xL, and Bid, which are potent inducers of apoptosis, the cleavage product of ?HV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-xL, however, was able to abrogate the cell death in BHK cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-xL overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation. During these studies, Bcl-xL was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-xL. However, even during such harsh treatment, Bcl-xL was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 555–564, 2000.

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-xL. In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclxL cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-xL also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclxL, and CHO-bclxL cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-xL in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 544–554, 2000.

Background:To assess the feasibility of intermittent androgen suppression in patients with metastatic prostate cancer and to quantify the improvement in the quality of life.Methods:Forty–three patients with M1 b prostate cancer were treated by intermittent hormonal deprivation using luteinizing hormone–releasing hormone (LHRH) analogue alone (n = 11), or associated with an antiandrogen (n = 32). The prospective nonrandomized study required an initial therapy period of 12 months with a stable biological response during 6 months (PSA, testosterone). Treatment was resumed when the serum PSA value recovered to 20 ng/ml, or when local failure or new bone metastasis occurred. The assessment of quality of life was carried out using the EORTC QLQ–C30.Results:The mean follow–up time was 43.7 months. After the initial 12 months of androgen suppression, one patient with a minimal disease was off–therapy with a follow–up of 18 months. For the 42 other patients, the mean off–therapy period was 6.7 months. In the second therapy period (9–12 months), 7 patients were hormono–independent and died with a mean survival time of 27 months; 35 patients were responders. The mean off–therapy length in the second cycle was short (3.8 months). After this time, androgen suppression therapy was reintroduced permanently, but 10 patients were hormono–independent. No difference was observed in the EORTC QLQ–C30 between therapy and off–therapy periods, only a rapid decrease in adverse events due to the hormonal deprivation was reported in all cases during the off–therapy period.Conclusions:Intermittent androgen suppression in patients with M1 b prostate cancer could be associated with a significant period off–therapy in the first cycle (55.8%), and with a chance of second hormone response. But in the second cycle, the off–therapy period length was short and required a careful follow–up.

We report that mutation of specific residues in the human B2 bradykinin (BK) receptor induces its marked constitutive activation, evaluated through inositol phosphate production in COS-7 cells expressing the wild-type or mutant receptors. We provide evidence for a strikingly high constitutive activation of the B2 receptor induced by alanine substitution of the Asn113 residue, located in the third transmembrane domain. These results are reminiscent of our previous finding that mutation of the homologous Asn111 residue induces constitutive activation of the AT1 angiotensin II receptor. BK overstimulation of the constitutively activated mutant N113A receptor was also observed. Phe replacement of the Trp256 residue, fairly conserved in transmembrane domain VI of G protein-coupled receptors, also induced a less prominent but significant constitutive activation. Interestingly, the peptidic HOE 140 compound and an original nonpeptidic compound LF 16 0335, which both behaved as inverse agonists of the wild-type receptor expressed in COS-7 cells, became potent and efficient agonists of the two constitutively activated mutant N113A and W256F receptors. These parallel changes observed for two chemically unrelated series can serve as a basis for future studies of structure-function relationships and modeling of activation processes, based on a detailed analysis of the network of helix-helix interactions, which stabilize the inactive receptor conformation and undergo rearrangements on transition to activated states.

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 298–305, 1999.

Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Cη (PKCη). PKCη transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCη were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCη. We found that PKCη is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCη cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCη results in cell cycle arrest at the G1/S transition. These results suggest that the expression and proteolytic activation of PKCη play an important role in the regulation of cell division and cell death during early B-cell development.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome cinto the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation.

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O⨪2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O⨪2levels appears to activate or modulate cell death, as a recombinant SV expressing the O⨪2scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O⨪2rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O⨪2by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.

Phytochrome is the red/far-red absorbing photoreceptor active in photomorphogenesis, the apoprotein of which is encoded by a small gene family (PHYA, PHYB, PHYC, PHYD and PHYE). A novel phytochrome B-deficient mutant, phyB-103, was isolated from a screen of EMS-mutagenised Arabidopsis M2 seed. phyB-103 carries a G-to-A base substitution at the 5' splice site +1 G nucleotide of intron 1 of PHYB. The phyB-103 PHYB transcript is larger than the wild-type PHYB transcript and DNA sequence analysis showed that the entire intron is retained in the mature PHYB transcript of phyB-103. Thus the phyB-103 G-to-A substitution prevents intron splicing. The retained intron contains within it an in-frame stop codon, and the predicted PHYB-103 apoprotein thus terminates prematurely. phyB-103 is therefore likely to be a null allele of PHYB, consistent with the observation that the phenotype conferred by phyB-103 is as severe as that conferred by previously described phyB null alleles.

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild‐type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho‐cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla‐vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra‐violet‐B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul‐traviolet‐B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.

Abstract: Transformed plants from three cultivars of Eustoma grandiflorum (lisianthus) were produced by cocultivating young leaf pieces with Agrobacterium tumefaciens strain A722 containing the binary vectors pKIWI110 and pLN26. Both vectors contain the selectable marker gene for neomycin phosphotransferase II. pKIWI110 also contains the reporter gene for β-d-glucuronidase, and pLN26, the chalcone synthase antisense gene. Southern DNA analysis revealed that all the kanamycin-resistant transformants tested contained copies of the transgenes integrated in their genome. The two plants transformed with pKIWI110 show β-d-glucuronidase expression in their mature leaves and selected transformants passed on the kanamycin-resistant phenotype to the F1 generation.

Viruses have exploited a fascinating array of mechanisms to inhibit programed cell death. Different viruses tamper with cell suicide pathways by terminating cell death receptor signalling, mimicking cellular Bc1-2 functions, encoding protease inhibitors and other tactics. These stealth strategies are likely to be essential for the preservation of some viruses as it appears nearly impossible for a virus to enter a cell and set up housekeeping without setting off the alarm system that triggers activation of the cell death pathway. Thus, many viruses have developed strategies to subvert the cellular selfdestruct mechanisms, thereby providing suitable environments for long-term persistence, the establishment of latency or for progeny virus production. Investigation of these viral strategies has significantly advanced our knowledge of cellular death pathways and has impacted on our understanding of viral pathogenesis.Copyright 1998 Academic Press

ABSTRACTSindbis 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.

OBJECTIVE: Many cases of porphyria cutanea tarda have been described in association with human immunodeficiency virus (HIV) infection in young individuals. The link between hepatitis C virus (HCV) and porphyria cutanea tarda is even stronger as more than 50% of patients who have this diagnosis in Italy, France, or Spain are also infected by HCV. To study the role of viral infections on the metabolism of porphyrins, we measured the urinary porphyrin levels in patients with HIV and HCV infections. DESIGN: Survey; prospective study. SETTING: University Hospital of Strasbourg, France. PATIENTS: Sixty-one HIV-positive patients, 56 HCV-positive patients, 60 HIV- and HCV-positive patients, and 51 HIV- and HCV-negative control subjects were randomly selected. None had clinical signs of porphyria or a familial history of porphyria. MAIN OUTCOME MEASURES: The porphyrin-excretion profile was determined by high-performance liquid chromatography on fresh urine samples. The HIV and HCV viremias were quantified in the serum by the branched DNA assay. Measures were planned before data collection began. RESULTS: The porphyrin-excretion profile typical of porphyria cutanea tarda was found in only 1 of 177 patients. In the remaining 176 patients, the mean coproporphyrin level was significantly raised in HCV-positive patients and even higher in patients who were HIV- and HCV-positive. The coproporphyrin level was not correlated to the alanine aminotransferase level, the CD4+ cell count, or the HCV and HIV viremias. CONCLUSIONS: In cases of infection with HIV, HCV, or both, the development of a porphyria cutanea tarda urinary profile is a rare event (0.56% in this study), but coproporphyrin excretion is increased. This could be related to hepatic changes induced by the viruses. Our results do not support the hypothesis of a direct viral effect on the porphyrin metabolism. Infection with HIV, HCV, or both may be a major triggering factor, but is not sufficient to induce porphyria.Arch Dermatol. 1996;132:1448-1452

A new method for centering and reorienting automatically the left ventricle in thallium-201 myocardial single photon emission computed tomography (SPET) is proposed. The processing involves the following steps: (a) the transverse sections of the left ventricle are segmented, (b) the three-dimensional skeleton of the left ventricle is extracted using tools of mathematical morphology, (c) the skeleton is fitted to a quadratic surface by the least-squares method, (d) the left ventricle is reoriented and centered using the long axis and the coordinates of the centre of the quadratic surface. A series of 30 consecutive exercise and redistribution 201T1 SPET studies were centered and reoriented by two operators twice with this method, and twice manually. There was no significant difference in the mean realignment performed by the automatic and the manual methods while centering differed moderately in some instances. In all cases and for all parameters, the reproducibility of the automatic method was 1.00, while it ranged between 0.74 and 0.98 with the manual centering and reorientation. This automatic approach provides a fast and highly reproducible method for the reconstruction of short- and long-axis sections of the left ventricle in 201T1 SPET.

Abstract: To test the hypothesis that the phenotypic abnormalities seen in cases with apparently balanced chromosomal rearrangements are the result of the presence of cryptic deletions or duplications of chromosomal material near the breakpoints, we analyzed three cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities. We characterized the breakpoints in these cases by using microsatellite analysis by polymerase chain reaction and fluorescence in situ hybridization analysis of yeast artificial chromosome clones selected from the breakpoint regions. Molecular characterization of the translocation breakpoint in patient 1 [46,XY,t(2;6)(p22.2;q23.1)] showed the presence of a 4- to 6-Mb cryptic deletion between markers D6S412 and D6S1705 near the 6q23.1 breakpoint. Molecular characterization of the proximal inversion 7q22.1 breakpoint in patient 2 [46,XY,inv(7)(q22.1q32.1)] revealed the presence of a 4-Mb cryptic deletion between D7S651 and D7S515 markers. No deletion or duplication of chromosomal material was found near the breakpoints in patient 3 [46,XX,t(2;6)(q33.1;p12.2)]. Our study suggests that a systematic molecular study of breakpoints should be carried out in cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities, because cryptic deletions near the breakpoints may explain the phenotypic abnormalities in these cases.

The results of lymphocyte functional studies performed with the peripheral blood mononuclear cells (MNCs) of patient Wa with a non-Sezary T-cell leukemia are described. The aggressive clinical course and the distinctive histologic, cytochemical, and cytogenetic features of this T-cell lymphoproliferative disorder are detailed in an accompanying report. Immunologic marker analysis revealed that 76% of the Wa MNC fraction formed rosettes with sheep erythrocytes (E-rosettes), and on histologic examination, 95% of the E-rosettes contained neoplastic lymphocytes; 10% of the MNCs formed EAC rosettes; and 13% showed SIg positivity by immunofluorescence. In vitro mitogenic responses of Wa cells measured by 3H-thymidine incorporation were extremely low, and responses to Candida and streptococcal antigens were absent. Although a normal percentage of B cells was evident in the Wa MNC fraction, no Ig synthesis occurred in either pokeweed mitogen (PWM) stimulated or unstimulated cultures. In coculture experiments with control MNCs from two normal individuals, the Wa cells exhibited no suppressor or helper effects on the normal PWM-stimulated Ig synthesis. Importantly however, the Wa cells demonstrated suppressor effects on the normal mixed lymphocyte reaction (MLR) occurring between populations of allogeneic MNCs from two normal donors. The degree of MLR suppression increased with the presence of greater numbers of Wa cells, suggesting a dose-response relationship in these three-way MLRs. Control three-way MLRs that included two normal allogeneic lymphoid populations and other neoplastic T-cell suspensions, unrelated to Wa cells, did not show any MLR suppressor effects. Unlike the Con-A-induced MLR suppression mediated by a subset of normal T suppressor cells, the Wa suppression did not require preincubation with mitogen for the expression of this MLR suppressor activity. The absence of detectable suppressor effects by Wa cells on the proliferative responses of normal lymphocytes to Con-A and in the Ig synthesis of normal MNCs suggested that the Wa MLR suppression was a specific immune activity of the neoplastic cells rather than a nonspecific toxic effect. These data demonstrating MLR suppressor activity without suppressor effects on normal PWM-stimulated Ig synthesis have described a selective suppressor capacity possessed by the Wa cells and emphasize the usefulness of employing multiple assay systems in testing neoplastic T cells for putative T- and B-cell effector functions. Moreover, the results of this combined analysis of histology, cytochemistry, cytogenetics, and immune activity of these neoplastic T cells should provide a basis for future comparisons of other T-cell lymphoproliferative disorders with definable lymphocyte function.

Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neocells, which were all dead by three days post-infection. Though infected AT3-neocells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.

This question of whether fungi undergo apoptosis-like programmed cell death can be separated into two questions. One question is about applying the term “apoptosis” to fungi, and the other is a more challenging question of whether fungi have evolved mechanisms that inflict self-injury.

Comment on: Zou S, et al. Cell Cycle 2011; 10: 989-98.

Several letters to the editor are presented in response to the articles in previous issues which includes article on Islay and Jura in Scotland, Kingsbarns Distillery, and concern over wind farming in Scotland.

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