Biomedical subjects

M Bushell

Publications and source records attributed to M Bushell.

17 recordsLinked to original sources

PubMed2006

TOPs and their regulation.

Upon cell-cycle arrest or nutrient deprivation, the cellular rate of ribosome production is reduced significantly. In mammalian cells, this effect is achieved in part through a co-ordinated inhibition of RP (ribosomal protein) synthesis. More specifically, translation initiation on RP mRNAs is inhibited. Translational regulation of RP synthesis is dependent on cis-elements within the 5'-UTRs (5'-untranslated regions) of the RP mRNAs. In particular, a highly conserved 5'-TOP (5'-terminal oligopyrimidine tract) appears to play a key role in the regulation of RP mRNA translation. This article explores recent developments in our understanding of the mechanism of TOP mRNA regulation, focusing on upstream signalling pathways and trans-acting factors, and highlighting some interesting observations which have come to light following the recent development of cDNA microarray technology coupled with polysome analysis.

5' Untranslated Regions↗

PubMed2005

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors.

During apoptosis, there is a reduction in translation initiation caused by caspase cleavage of several of the factors required for the cap-dependent scanning mechanism. Under these circumstances, many proteins that are required for apoptosis are instead translated by the alternative method of internal ribosome entry. This mechanism requires the formation of a complex RNA structural element and in the presence of internal ribosome entry segment (IRES)-trans-acting factors (ITAFs), the ribosome is recruited to the RNA. The interactions of several ITAFs with IRESs have been investigated in detail, and several mechanisms of action have been noted, including acting as chaperones, stabilising and remodelling the RNA structure. Structural remodelling by PTB in particular will be discussed, and how this protein is able to facilitate recruitment of the ribosome to several IRESs by causing previously occluded sites to become more accessible.

Apoptosis↗

PubMed2004

Translation inhibition during the induction of apoptosis: RNA or protein degradation?

The induction of apoptosis leads to a substantial inhibition of protein synthesis. During this process changes to the translation-initiation factors, the ribosome and the cellular level of mRNA have been documented. However, it is by no means clear which of these events are necessary to achieve translational shutdown. In this article, we discuss modifications to the translational apparatus that occur during apoptosis and examine the potential contributions that they make to the inhibition of protein synthesis. Moreover, we present evidence that suggests that a global increase in the rate of mRNA degradation occurs before the caspase-dependent cleavage of initiation factors. Increased mRNA decay is temporally correlated with the shutdown of translation and therefore plays a major role in the inhibition of protein synthesis in apoptotic cells.

Apoptosis↗

PubMed2001

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.

3C Viral Proteases↗

PubMed2000

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.

Apoptosis↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Apoptosis↗

PubMed2000

Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis.

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.

Apoptosis↗

PubMed1998

Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines.

We have investigated the effect of inducing apoptosis in BJAB and Jurkat cells on the cellular content of several polypeptide chain initiation factors. Serum deprivation results in inhibition of protein synthesis and induction of apoptosis in BJAB cells; at early times, there is selective degradation of polypeptide initiation factor eIF4G but no major losses of other key initiation factors. The disappearance of full length eIF4G is accompanied by the appearance of smaller forms of the protein, including a major product of approximately 76 kDa. Apoptosis induced by cycloheximide results in similar effects. Both total cytoplasmic eIF4G and eIF4G associated with eIF4E are degraded with a half-life of 2-4 h under these conditions. Treatment of serum-starved or cycloheximide-treated cells with Z-VAD.FMK or Z-DEVD.FMK, which inhibit caspases required for apoptosis, protects eIF4G from degradation and blocks the appearance of the ca. 76 kDa product. Exposure of BJAB cells to rapamycin rapidly inhibits protein synthesis but does not lead to acute degradation of eIF4G. In both BJAB and Jurkat cells induction of apoptosis with anti-Fas antibody or etoposide also results in the selective loss of eIF4G, which is inhibitable by Z-VAD.FMK. These data suggest that eIF4G is selectively targeted for cleavage as cells undergo apoptosis and is a substrate for proteases activated during this process.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Cleavage of translation initiation factor 4G (eIF4G) during anti-Fas IgM-induced apoptosis does not require signalling through the p38 mitogen-activated protein (MAP) kinase.

Initiation factor (eIF) 4G plays a key role in the regulation of translation, acting as a bridge between eIF4E and eIF3, to allow an mRNA molecule to associate with the 40S ribosomal subunit. In this study, we show that activation of the Fas/CD95 receptor complex in Jurkat cells induces the degradation of eIF4G, the inhibition of total protein synthesis and cell death. These responses were prevented by the caspase inhibitors, zVAD.FMK and zDEVD.FMK. We also show that, in contrast to Saccharomyces cerevisiae, although rapamycin caused a modest inhibition of protein synthesis it did not induce apoptosis or the cleavage of eIF4G. Studies with the specific inhibitor, SB203580, have shown that signalling through the p38 MAP kinase pathway is not required for either the Fas/CD95-induced cleavage of eIF4G or cell death. These data suggest that the cleavage of eIF4G and the inhibition of translation play an integral role in Fas/CD95-induced cell death in Jurkat cells.

Amino Acid Chloromethyl Ketones↗

PubMed1987

Management of diffuse peritonitis by prolonged postoperative peritoneal lavage.

Prolonged postoperative peritoneal lavage has been used as a part of the management of 55 patients with diffuse peritonitis. The lavage technique consisted of 60 min cycles of instillation and drainage of a lavage fluid into the peritoneal cavity via a peritoneal dialysis catheter. The lavage fluid was a slightly hypertonic salt solution containing antibiotics, usually cefotaxime and metronidazole. The overall mortality rate in the series was 11% (6/55). Only one of these patients had residual abdominal sepsis present at post-mortem, the remaining deaths being due to a progression of the pre-existing disease. Five patients showed evidence of further intra-abdominal sepsis. In three of these patients this was associated with the presence of a previously well established abscess cavity. The overall results indicate that, for this group of patients recognized to be at high risk of mortality or further sepsis, the use of prolonged postoperative peritoneal lavage is associated with an improved outcome.

Adolescent↗

PubMed1986

Gallbladder emptying in man related to fasting duodenal migrating motor contractions.

Animal studies have shown that the gallbladder (GB) empties partially during fasting in relation to phase II of the interdigestive motor cycle (IDMC). It has been assumed that in man the GB remains inert during fasting, although there have been no studies in which repeated sequential measurements of gallbladder volume have been made and related to the IDMC. In this study the possibility of linkage between the IDMC and GB volume has been examined in nine fasting human volunteers (five males/four females). A multilumen constantly perfused manometry catheter was positioned in the duodenum and motor activity recorded continuously. Phase III of the IDMC was characterized by phasic contractions, with a frequency of 10-12/min for at least 2 min, which had a distal propagative pattern and were followed by a motor quiescence (Phase I). GB volume was calculated at 15 min intervals from ultrasound measurements of the maximal length and diameter of the GB by the summation of cylinders method, and related to the time of onset of each phase III of the IDMC. In all nine subjects GB volume decreased prior to the onset of phase III and increased following its passage (P less than 0.01). GB volume decrease ranged between 6 and 56% of maximal GB volume (median 18%) and subsequent filling ranged from 14 to 53% (median 27%). The maximal volume decrease occurred 30 min prior to the onset of phase III (P less than 0.02). These studies have demonstrated that the human GB empties partially during phase II of the IDMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1986

Peroperative sphincter of Oddi manometry: motility disorder in patients with cholelithiasis.

Intraluminal pressure recordings from the sphincter of Oddi (SO) have made significant contributions towards the understanding of normal SO function and are being used for the diagnosis of SO motility abnormalities. In this study the endoscopic intraluminal methods for measuring SO pressure changes have been adapted for use under sterile conditions during surgery on the biliary system. Sphincter of Oddi pressure measurement in a group of patients undergoing elective cholecystectomy for gallstones, were compared with a group of control subjects undergoing endoscopic study of the SO. There was no significant difference in CBD pressure. SO basal pressure, SO wave amplitude and SO wave frequency. However, a highly significant difference was noted in the propagation direction of the SO contractions. The control subjects had a predominance of antegrade contractions whereas patients undergoing cholecystectomy had a predominance of retrograde contractions. This result suggests an association between SO motility disorder and the presence of gallstones.

Adult↗

PubMed1982

Acid-base imbalance and ulceration in the cold restrained rat.

Using the cold restrained rat model of stress ulceration, we have examined the influence of metabolic acidosis, metabolic alkalosis, and respiratory acidosis on the development of gastric erosions. The rats were restrained in tightly fitting perspex chambers at 6 degrees C for 3 hours. Acid-base imbalance was achieved by infusion of NH4Cl or NaHCO3 or by exposure to 5% CO2. The degree of ulceration was expressed by a lesion score of 0 to 4. The control group showed a score of 2.5 +/- 0.2 (mean +/- SEM). With metabolic acidosis the score was 3.6 +/- 0.2, and with metabolic alkalosis the score was 0.9 +/- 0.4. Both values were significantly different from control values (P less than 0.005). Respiratory acidosis was associated with a score similar to that of the control group. The values obtained appeared to be independent of gastric luminal acidity. The findings indicate that the systemic HCO-3 concentration is a significant determinant of the degree of ulceration in the cold restrained rat.

Acid-Base Imbalance↗

PubMed1980

Role of acid-base status in the response of the isolated amphibian gastric mucosa to back diffusion of H+.

The effect of a period of induced back diffusion of H+ on the isolated amphibian gastric mucosa was studied under various conditions. Under standard conditions (HCO3--buffered nutrient solution; 95% O2-5% CO2), passage of an electrical current of 500 microA/cm2 for 15 min from a secretory fluid of pH 2.10 across the mucosal resulted in a decrease of the transmucosal potential difference (PD) of 10.9 +/- 1.9 mV, a decrease of resistance (R) of 48 +/- 26 ogema cm2 and a decrease of short circuit current (Isc) of 18 +/- 7 microA/cm2. Flux of the neutral molecule, erythritol from secretory (S) to nutrient (N) fluid increased by 66% and the active transport of Cl- N lead to S decreased from 2.9 to 1.9 microeq/cm2/hr. With removal of HCO3- from the nutrient fluid and with inhibition of carbonic anhydrase activity the period of back diffusion induced significantly greater change in the electrical measurements than occurred when under standard conditions. This increased effect of back diffusion of H+ was not changed by change of the nutrient fluid pH from 7.20 to either 6.6 or 8.2. Increase of the HCO3- concentration of the nutrient fluid to 35 mM or decrease of the CO2 content of the aerating gas to 1% were associated with significantly less change of the electrical measurements than occurred with standard conditions. These studies support the proposal that the the neutralizing reaction HCO3- + H+ leads to CO2 + H2O plays a central role in the gastric mucosal handling of backing diffusing H+ and suggests that the capacity and poise of the HCO3-/CO2 buffer system is an important determinant of the ability of the mucosa to tolerate luminal acidity.

Acid-Base Equilibrium↗

PubMedDate not supplied

Gall-bladder and sphincter of Oddi response to cholecystokinin in the Australian possum.

The effects of cholecystokinin octapeptide (CCK-OP) on the gall-bladder (GB) and sphincter of Oddi (SO) of the Australian brush tailed possum were examined in 45 anaesthetized animals. CCK-OP (20-640 ng/kg) consistently caused the GB to contract in a dose-dependent manner (Kruskal Wallis P less than 0.05). In 20 animals, the same dose range of CCK-OP produced an excitatory response in the SO, increasing the SO motility index (MI = frequency of contractions x mean peak amplitude) dose-dependently (Kruskal Wallis P less than 0.05). In five animals, an inhibitory response, that is, a decrease in MI, was recorded, with 640 ng/kg of CCK-OP producing a 50% decrease in MI. In the remaining 20 animals, variable responses of both excitation and inhibition were elicited within the same animal. The action of CCK-OP on the SO and GB was not modified by atropine, phentolamine or propranolol. Tetrodotoxin (TTX) reversed the inhibitory responses of the SO to CCK-OP such that responses were excitatory (sign test P less than 0.05). TTX did not alter the response of the GB to CCK-OP. It is concluded that CCK-OP acts directly on smooth muscle receptors of the GB. In the SO, its action is mediated via non-cholinergic, non-noradrenergic inhibitory neurons and also by a direct excitatory action on the smooth muscle of the SO.

Animals↗