An efficient and cost-effective method of generating postnatal (P2-5) mouse primary hippocampal neuronal cultures.

BACKGROUND: Primary culture of postnatal central neurons is a widely used methodology for applications such as the investigation of neuronal development, protein trafficking/distribution and cellular signalling. However, successful production and maintenance of such cultures, particularly from postnatal animals, can be challenging. In attempting to surmount these difficulties, several disparate culturing methodologies have been developed. Such methodologies are centred on the identification and optimisation of critical steps and, as such, the protocols and reagents utilised can differ quite markedly from protocol to protocol, often with the suggestion that the use of a (usually expensive) proprietary reagent(s), lengthy substrate preparation and/or cell isolation techniques is/are necessary for successful culture preparation. NEW METHOD: Herein, we present a simple and inexpensive protocol for the preparation of primary hippocampal neurons from postnatal (2-5 day old) mice, which remain viable for experimental use for over one month. RESULTS: Neurons cultured using this method follow well established developmental norms and display typical responses to standard physiological stimuli such as depolarisation and certain pharmacological agents. COMPARISON WITH EXISTING METHODS/CONCLUSION: By using a novel trituration technique, simplified methodology and non-proprietary reagents, we have developed a reliable protocol that enables the cost effective and efficient production of high quality postnatal mouse hippocampal cultures. This method, if required, can also be utilised to prepare neurons both from other regions of the brain as well as from other species such as rat.

Psychological approaches to chronic pain management: evidence and challenges.

Psychological interventions are a mainstay of modern pain management practice and a recommended feature of a modern pain treatment service. Systematic reviews for the evidence of psychological interventions are reviewed in this article. The evidence for effectiveness is strongest for cognitive behavioural therapy with a focus on cognitive coping strategies and behavioural rehearsal. Most evidence is available for treatments of adult pain, although adolescent chronic pain treatments are also reviewed. It is clear that treatment benefit can be achieved with cognitive behavioural methods. It is possible to effect change in pain, mood, and disability, changes not achieved by chance or by exposure to any other treatment. However, the overall effect sizes of treatments for adults, across all trials, are modest. Reasons for the relatively modest treatment effects are discussed within the context of all treatments for chronic pain being disappointing when measured by the average. Suggestions for improving both trials and evidence summaries are made. Finally, consideration is given to what can be achieved by the pain specialist without access to specialist psychology resource.

Immunohistochemical mapping of total and phosphorylated eukaryotic initiation factor 4G in rat hippocampus following global brain ischemia and reperfusion.

Partial proteolysis and phosphorylation of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) occur in reperfused brain, but the contribution of eIF4G alterations to brain injury has not been established. A component of the complex delivering mRNA to the small ribosomal subunit, eIF4G is also found in stress granules. Stress granules sequester inactive 48S preinitiation complexes during stress-induced translation arrest. We performed double-labeling immunofluorescence histochemistry for total or ser 1108 phosphorylated eIF4G and the stress granule component T-cell internal antigen following normothermic, 10 min cardiac arrest-induced global brain ischemia and up to 4 h reperfusion in the rat. In cornu ammonis (Ammon's horn; CA) 1 at 90 min and 4 h reperfusion, eIF4G staining transformed from a homogeneous to an aggregated distribution. The number of eIF4G-containing stress granules differed between CA1 and CA3 during reperfusion. In hippocampal pyramidal neurons, phosphorylated eIF4G appeared exclusively in stress granules. Supragranular interneurons of the dentate gyrus showed a large increase in cytoplasmic eIF4G(P) following reperfusion. Immunoblot analysis with antisera against different portions of eIF4G showed a large increase in phosphorylated C-terminal eIF4G fragments, suggesting these accumulate in the cytoplasm of dentate gyrus interneurons. Thus, altered eIF4G subcellular compartmentalization may contribute to prolonged translation arrest in CA1 pyramidal neurons. Accumulation of phosphorylated eIF4G fragments may contribute to the vulnerability of dentate interneurons. Ischemia and reperfusion invoke different translational control responses in distinct hippocampal neuron populations, which may contribute to the differential ischemic vulnerabilities of these cells.

Initiation factor modifications in the preapoptotic phase.

Recent studies have identified several mechanistic links between the regulation of translation and the process of apoptosis. Rates of protein synthesis are controlled by a wide range of agents that induce cell death, and in many instances, the changes that occur to the translational machinery precede overt apoptosis and loss of cell viability. The two principal ways in which factors required for translational activity are modified prior to and during apoptosis involve (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. In this review, we summarise the principal targets for such regulation, with particular emphasis on polypeptide chain initiation factors eIF2 and eIF4G and the eIF4E-binding proteins. We indicate how the functions of these factors and of other proteins with which they interact may be altered as a result of activation of apoptosis and we discuss the potential significance of such changes for translational control and cell growth regulation.

Behavioural treatment for chronic low-back pain.

BACKGROUND: Behavioural treatment, commonly used in the treatment of chronic low-back pain (CLBP), is primarily focused at reducing disability through the modification of environmental contingencies and cognitive processes. In general, three behavioural treatment approaches are distinguished: operant, cognitive and respondent. OBJECTIVES: To determine if behavioural therapy is more effective than reference treatments for CLBP, and which type of behavioural treatment is most effective. SEARCH STRATEGY: We searched the CENTRAL, MEDLINE, EMBASE, and PsycLIT databases up to October 2003. References of identified randomised trials and relevant systematic reviews were screened. SELECTION CRITERIA: Only randomised trials on behavioural treatment for non-specific CLBP were included. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed the methodological quality and extracted the data. The magnitude of effect was assessed by computing a pooled effect size for post-treatment and long-term results for each comparison, for each domain (i.e., behavioural outcomes, overall improvement, back pain specific and generic functional status, return to work, and pain intensity) using the random effects model. MAIN RESULTS: Seven studies (33%) were considered high quality. Comparing behavioural treatment to waiting list control (WLC) revealed strong evidence (4 trials, 134 people) in favour of a combined respondent-cognitive therapy for a medium positive effect on pain, and moderate evidence (2 trials, 39 people) in favour of progressive relaxation for a large positive effect on pain and behavioural outcomes (short-term only). When comparing operant treatment to WLC no significant differences could be detected on general functional status (strong evidence: 2 trials, 87 people) or on behavioural outcomes (moderate evidence; 3 trials, 153 people) (short-term only). There is limited evidence (1 trial, 98 people) that a graded activity program in an industrial setting is more effective than usual care for early return to work and reduced long-term sick leave. There is limited evidence (1 trail, 39 people) that there are no differences between behavioural treatment and exercises. Finally, there is moderate evidence (6 trials, 210 people) that there are no significant differences in short-term and long-term effectiveness when behavioural components are added to usual treatment programs for CLBP (i.e. physiotherapy, back education) on pain, generic functional status and behavioural outcomes. AUTHORS' CONCLUSIONS: Combined respondent-cognitive therapy and progressive relaxation therapy are more effective than WLC on short-term pain relief. However, it is unknown whether these results sustain in the long term. No significant differences could be detected between behavioural treatment and exercise therapy. Whether clinicians should refer patients with CLBP to behavioural treatment programs or to active conservative treatment cannot be concluded from this review.

Toxicity of cadmium and zinc to the decaudised cercarial life-span of Diplostomum spathaceum (Trematoda: Diplostomidae).

The toxicity of cadmium and zinc at concentrations ranging from 0.1 to 10000 microg/l to the life-span of decaudised cercarial bodies (cercariae that have shed their tails) of Diplostomum spathaceum was investigated. The effects of metal exposure at 3 temperatures (12, 20, and 25 degrees C) and 3 levels of water hardness (distilled water, soft water and hard water) were studied. In general, under most experimental conditions increasing metal concentrations reduced the life-span of decaudised cercariae. Increasing water hardness and decreasing water temperature caused an increase in the life-span of both control and metal exposed decaudised cercariae. However, at certain isolated metal concentrations, associated with a specific level of water hardness and temperature, increased survival above controls occurred. Differences in the relative toxicity of cadmium and zinc were dependent on the environmental conditions of exposure. The decaudised cercarial life-span under metal exposure was found to be generally independent of the overall cercarial survival and tail loss in most experimental conditions. Prolonged exposure to cadmium and zinc caused changes in the decaudised cercarial life-span when compared to individuals decaudised during the initial 24 h exposure period to those which were decaudised during the final 24 h period of cercarial survival. The validity of studying the decaudised cercarial life-span as an indicator of 'fitness' of larvae to migrate through the tissues of the target fish host, in terms of glycogen utilization, was assessed for those cercariae decaudised during the initial 24 h exposure period only. A limited reduction in the decaudised cercarial life-span during this period compared to controls was recorded, which may possibly indicate a reduced penetration 'fitness' of cercariae exposed to cadmium and zinc. The importance and relevance of these findings to parasite migration and establishment in the fish host are discussed.

Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.

Proteasome inhibitors and immunosuppressive drugs promote the cleavage of eIF4GI and eIF4GII by caspase-8-independent mechanisms in Jurkat T cell lines.

Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti-Fas-mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild-type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase-8 and caspase-3-like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase-3-like activity in a caspase-8-deficient Jurkat cell line which is resistant to anti-Fas-mediated apoptosis. Using specific anti-peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti-Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti-Fas antiserum. These data suggest that caspase-8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells.

Truncated initiation factor eIF4G lacking an eIF4E binding site can support capped mRNA translation.

Picornavirus proteases cleave translation initiation factor eIF4G into a C-terminal two-thirds fragment (hereafter named p100) and an N-terminal one-third fragment, which interacts with the cap-binding factor eIF4E. As the timing of this cleavage correlates broadly with the shut-off of host cell protein synthesis in infected cells, a very widespread presumption has been that p100 cannot support capped mRNA translation. Through the use of an eIF4G-depleted reticulocyte lysate system, we show that this presumption is incorrect. Moreover, recombinant p100 can also reverse the inhibition of capped mRNA translation caused either by m7GpppG cap analogue, by 4E-BP1, which sequesters eIF4E and thus blocks its association with eIF4G, or by cleavage of endogenous eIF4G by picornavirus proteases. The concentration of p100 required for maximum translation of capped mRNAs is approximately 4-fold higher than the endogenous eIF4G concentration in reticulocyte lysates. Our results imply that picornavirus-induced shut-off is not due to an intrinsic inability of p100 to support capped mRNA translation, but to the viral RNA outcompeting host cell mRNA for the limiting concentration of p100.

Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G.

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m(7) guanosine cap.

In eukaryotes the majority of mRNAs have an m(7)G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.

Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages.

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding protein in vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.

Behavioral treatment for chronic low back pain: a systematic review within the framework of the Cochrane Back Review Group.

STUDY DESIGN: A systematic review of randomized controlled trials. SUMMARY OF BACKGROUND DATA: The treatment of chronic low back pain is not primarily focused on removing an underlying organic disease but at the reduction of disability through the modification of environmental contingencies and cognitive processes. Behavioral interventions are commonly used in the treatment of chronic (disabling) low back pain. OBJECTIVES: To determine whether behavioral therapy is more effective than reference treatments for chronic nonspecific low back pain and which type of behavioral treatment is most effective. METHODS: The authors searched the Medline and PsychLit databases and the Cochrane Controlled Trials Register up to April 1999, and Embase up to September 1999. Also screened were references of identified randomized trials and relevant systematic reviews. Methodologic quality assessment and data extraction were performed independently by two reviewers. The magnitude of effect was assessed by computing a pooled effect size for each domain (i.e., behavioral outcomes, overall improvement, back pain-specific and generic functional status, return to work, and pain intensity) using the random effects model. RESULTS: Only six (25%) studies were high quality. There is strong evidence (level 1) that behavioral treatment has a moderate positive effect on pain intensity (pooled effect size 0.62; 95% confidence interval [CI] 0.25, 0.98), and small positive effects on generic functional status (pooled effect size 0.35; 95% CI: -0.04, 0.74) and behavioral outcomes (pooled effect size 0.40; 95% CI: 0.10, 0.70) of patients with chronic low back pain when compared with waiting-list controls or no treatment. There is moderate evidence (level 2) that a addition of behavioral component to a usual treatment program for chronic low backpain has no positive short-term effect on generic functional status (pooled effect size 0.31; 95% CI: -0.01, 0.64), pain intensity (pooled effect size 0.03; 95% CI:-0.30, 0.36), and behavioral outcomes (pooled effect size 0.19; 95% CI: -0.08, 0.45). CONCLUSIONS: Behavioral treatment seems to be an effective treatment for patients with chronic low back pain,but it is still unknown what type of patients benefit most from what type of behavioral treatment.

Utilizing the peptidyl-prolyl cis-trans isomerase pin1 as a probe of its phosphorylated target proteins. Examples of binding to nuclear proteins in a human kidney cell line and to tau in Alzheimer's diseased brain.

The human parvulin Pin1 is a member of the peptidyl-prolyl cis-trans isomerase group of proteins, which modulate the assembly, folding, activity, and transport of essential cellular proteins. Pin1 is a mitotic regulator interacting with a range of proteins that are phosphorylated before cell division. In addition, an involvement of Pin1 in the tau-related neurodegenerative brain disorders has recently been shown. In this context, Pin1 becomes depleted from the nucleus in Alzheimer's disease (AD) neurons when it is redirected to the large amounts of hyperphosphorylated tau associated with the neurofibrillary tangles. This depletion from the nucleus may ultimately contribute to neuron cell death. Recently we have devised a novel methodology in which exogenous Pin1 is used as a TEM probe for its target proteins. Here we extend this methodology to provide further evidence that Pin1 binds at enhanced levels to mitotic nuclear proteins and to hyperphosphorylated tau in AD brain. We suggest that exogenous Pin1 labeling can be used to elucidate the phosphorylation status of its target proteins in general and could specifically provide important insights into the development of tau-related neurodegenerative brain disorders.

Behavioral treatment for chronic low back pain: a systematic review within the framework of the Cochrane Back Review Group.

STUDY DESIGN: A systematic review of randomized controlled trials. SUMMARY OF BACKGROUND DATA: The treatment of chronic low back pain is not primarily focused on removing an underlying organic disease but at the reduction of disability through the modification of environmental contingencies and cognitive processes. Behavioral interventions are commonly used in the treatment of chronic (disabling) low back pain. OBJECTIVES: To determine whether behavioral therapy is more effective than reference treatments for chronic nonspecific low back pain and which type of behavioral treatment is most effective. METHODS: The authors searched the Medline and PsychLit databases and the Cochrane Controlled Trials Register up to April 1999, and Embase up to September 1999. Also screened were references of identified randomized trials and relevant systematic reviews. Methodologic quality assessment and data extraction were performed independently by two reviewers. The magnitude of effect was assessed by computing a pooled effect size for each domain (i.e., behavioral outcomes, overall improvement, back pain-specific and generic functional status, return to work, and pain intensity) using the random effects model. RESULTS: Only six (25%) studies were high quality. There is strong evidence (level 1) that behavioral treatment has a moderate positive effect on pain intensity (pooled effect size 0.62; 95% confidence interval [CI] 0. 25, 0.98), and small positive effects on generic functional status (pooled effect size 0.35; 95% CI: 0.04, 0.74) and behavioral outcomes (pooled effect size 0.40; 95% CI: 0.10, 0.70) of patients with chronic low back pain when compared with waiting-list controls or no treatment. There is moderate evidence (level 2) that a addition of behavioral component to a usual treatment program for chronic low backpain has no positive short-term effect on generic functional status (pooled effect size 0.31; 95% CI: 0.01, 0.64), pain intensity (pooled effect size 0.03; 95% CI: 0.30,0.36), and behavioral outcomes (pooled effect size 0.19; 95% CI: 0.08, 0.45). CONCLUSIONS: Behavioral treatment seems to be an effective treatment for patients with chronic low back pain,but it is still unknown what type of patients benefit most from what type of behavioral treatment.

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes.

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5'-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5'-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5'-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)(+) but not poly(A)(-) mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.

Differential requirements for caspase-8 activity in the mechanism of phosphorylation of eIF2alpha, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells.

Previously we have reported that induction of apoptosis in Jurkat cells results in an inhibition of overall protein synthesis with the selective and rapid cleavage of eukaryotic initiation factor (eIF) 4GI. For the cleavage of eIF4GI, caspase-3 activity is both necessary and sufficient in vivo, in a process which does not require signaling through the p38 MAP kinase pathway. We now show that activation of the Fas/CD95 receptor promotes an early, transient increase in the level of eIF2alpha phosphorylation, which is temporally correlated with the onset of the inhibition of translation. This is associated with a modest increase in the autophosphorylation of the protein kinase activated by double-stranded RNA. Using a Jurkat cell line that is deficient in caspase-8 and resistant to anti-Fas-induced apoptosis, we show that whilst the cleavage of eIF4GI is caspase-8-dependent, the enhancement of eIF2alpha phosphorylation does not require caspase-8 activity and occurs prior to the cleavage of eIF4GI. In addition, activation of the Fas/CD95 receptor results in the caspase-8-dependent dephosphorylation and degradation of p70(S6K), the enhanced binding of 4E-BP1 to eIF4E, and, at later times, the cleavage of eIF2alpha. These data suggest that apoptosis impinges upon the activity of several polypeptides which are central to the regulation of protein synthesis and that multiple signaling pathways are involved in vivo.

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.