Characterization of the PF-ILD phenotype in patients with advanced pulmonary sarcoidosis.

BACKGROUND: Advanced pulmonary sarcoidosis causes significant morbidity and can lead to death. Large trials demonstrated efficacy of antifibrotics in patients with progressive fibrosing interstitial lung diseases (PF-ILD), including a few with sarcoidosis. To date, little is known about this progressive fibrosing phenotype in sarcoidosis. Diffusion capacity of carbon monoxide (DLCO) may be a useful functional marker to screen for advanced pulmonary sarcoidosis. In this study, we describe a cohort with advanced pulmonary sarcoidosis and we gain insights in the progressive fibrosing phenotype in sarcoidosis. METHODS: Patients with sarcoidosis and a DLCO < 50% predicted were included in this retrospective cohort study. First measurement of DLCO < 50% predicted was the baseline. Lung function data, HRCT, pulmonary hypertension (PH) and mortality were collected. Patients with > 10% fibrosis on HRCT meeting the criteria for ILD-progression within 24 months were labelled as PF-ILD. With Cox-regression analysis predictors of mortality were established. RESULTS: 106 patients with a DLCO < 50% predicted were included. Evolution of forced vital capacity (FVC) varied widely between patients from - 34% to + 45% after 2 years follow-up, whereas change in DLCO varied between - 11% and + 26%. Fourteen patients (15%) met the PF-ILD criteria, of whom 6 (43%) died within 10 years versus 10 (13%) in the non PF-ILD group (p = 0.006). PH was present 12 (11%), 56 (53%) demonstrated > 10% fibrosis on HRCT. Independent predictors of mortality and lung transplantation in the whole cohort are PH, PF-ILD and UIP-like pattern. CONCLUSION: In conclusion, within this group with advanced pulmonary sarcoidosis disease course varied widely from great functional improvement to death. PF-ILD patients had higher mortality rate than the mortality in the overall pulmonary sarcoidosis group. Future research should focus on the addition of antifibrotics in these patients. Trial registration retrospectively registered.

Predicting local tumour progression after ablation for colorectal liver metastases: CT-based radiomics of the ablation zone.

PURPOSE: To assess whether CT-based radiomics of the ablation zone (AZ) can predict local tumour progression (LTP) after thermal ablation for colorectal liver metastases (CRLM). MATERIALS AND METHODS: Eighty-two patients with 127 CRLM were included. Radiomics features (with different filters) were extracted from the AZ and a 10 mm periablational rim (PAR)on portal-venous-phase CT up to 8 weeks after ablation. Multivariable stepwise Cox regression analyses were used to predict LTP based on clinical and radiomics features. Performance (concordance [c]-statistics) of the different models was compared and performance in an 'independent' dataset was approximated with bootstrapped leave-one-out-cross-validation (LOOCV). RESULTS: Thirty-three lesions (26 %) developed LTP. Median follow-up was 21 months (range 6-115). The combined model, a combination of clinical and radiomics features, included chemotherapy (HR 0.50, p = 0.024), cT-stage (HR 10.13, p = 0.016), lesion size (HR 1.11, p = <0.001), AZ_Skewness (HR 1.58, p = 0.016), AZ_Uniformity (HR 0.45, p = 0.002), PAR_Mean (HR 0.52, p = 0.008), PAR_Skewness (HR 1.67, p = 0.019) and PAR_Uniformity (HR 3.35, p < 0.001) as relevant predictors for LTP. The predictive performance of the combined model (after LOOCV) yielded a c-statistic of 0.78 (95 %CI 0.65-0.87), compared to the clinical or radiomics models only (c-statistic 0.74 (95 %CI 0.58-0.84) and 0.65 (95 %CI 0.52-0.83), respectively). CONCLUSION: Combining radiomics features with clinical features yielded a better performing prediction of LTP than radiomics only. CT-based radiomics of the AZ and PAR may have potential to aid in the prediction of LTP during follow-up in patients with CRLM.

Soil-test N recommendations augmented with PEST-optimized RZWQM simulations.

Improved understanding of year-to-year late-spring soil nitrate test (LSNT) variability could help make it more attractive to producers. We test the ability of the Root Zone Water Quality Model (RZWQM) to simulate watershed-scale variability due to the LSNT, and we use the optimized model to simulate long-term field N dynamics under related conditions. Autoregressive techniques and the automatic parameter calibration program PEST were used to show that RZWQM simulates significantly lower nitrate concentration in discharge from LSNT treatments compared with areas receiving fall N fertilizer applications within the tile-drained Walnut Creek, Iowa, watershed (>5 mg NL(-1) difference for the third year of the treatment, 1999). This result is similar to field-measured data from a paired watershed experiment. A statistical model we developed using RZWQM simulations from 1970 to 2005 shows that early-season precipitation and early-season temperature account for 90% of the interannual variation in LSNT-based fertilizer N rates. Long-term simulations with similar average N application rates for corn (Zea mays L.) (151 kg N ha(-1)) show annual average N loss in tile flow of 20.4, 22.2, and 27.3 kg N ha(-1) for LSNT, single spring, and single fall N applications. These results suggest that (i) RZWQM is a promising tool to accurately estimate the water quality effects of LSNT; (ii) the majority of N loss difference between LSNT and fall applications is because more N remains in the root zone for crop uptake; and (iii) year-to-year LSNT-based N rate differences are mainly due to variation in early-season precipitation and temperature.

Bone grafting, orthopaedic biomaterials, and the clinical need for bone engineering.

As the population ages, the number of operations performed on bone is expected to increase. Diseases such as arthritis, tumours, and trauma can lead to defects in the skeleton requiring an operation to replace or restore the lost bone. Surgeons can use autografts, allografts, and/or bone graft substitutes to restore areas of bone loss. Surgical implants are also used in addition or in isolation to replace the diseased bone. This review considers the application of available bone grafts in different clinical settings. It also discusses recently introduced bioactive biomaterials and highlights the clinical difficulties and technological deficiencies that exist in our current surgical practice.

Outcome of palliative esophageal stenting for malignant dysphagia: a retrospective analysis.

Greater than 50% of patients with esophageal carcinoma are found to be incurable at the time of diagnosis, leaving only palliative options. Self-expanding metal stents (SEMs) are effective for relieving symptoms and complications associated with esophageal carcinoma and improving quality of life. We undertook a retrospective analysis to evaluate the experience of palliative esophageal stenting for symptomatic malignant dysphagia in our institution over a period of 7 years. Between January 1999 and January 2006, 126 patients who received SEMs for malignant dysphagia were identified using an upper gastrointestinal specialist nurse clinician database. Data were obtained from patient case notes, endoscopy, histopathology, radiology, and external agency databases. Of the 126 identified, 36 patients were excluded from the analysis. A number of variables including age, sex, presenting complaints, type of stent, indications of stenting, success or failure of stent insertion, survival rate, and complication rate were analyzed. Of the 90 patients, 55 (61%) were male and 35 (39%) were female. The mean age of patients was 70.79 (range 40-97) years. The predominant presenting complaints were dysphagia (n = 81) and weight loss (n = 48). The indication for stenting was worsening dysphagia in all patients. Tumors were confined to the distal esophagus and esophagogastric junction in 73 patients (81%), and the mid-esophagus in 17 (19%). Adenocarcinoma was identified in 61 patients (67.8%) and squamous cell carcinoma in 29 (32.2%). Stenting numbers were comparable in endoscopic and radiologic groups (47 vs. 43), with successful stent deployment in 89 patients. The 7- and 30-day mortality was 9% (n = 8) and 28% (n = 25), respectively. Comparable numbers of early deaths were seen in both radiologic (n = 13) and endoscopic (n = 12) groups. Causes of early inpatient death included hemorrhage (n = 5), pneumonia (n = 7), exhaustion (n = 2), cardiac causes (n = 3), perforation (n = 1), and sepsis (n = 1). The number of patients with complications was 41 (45.6%), 25 in the surgical group and 15 in the radiologic group; the difference was not significant (P = 0.13). The mean survival time was 92.5 (0-638) days and median survival time was 61 days. A subgroup of patients with complete dysphagia (score 4) gained a mean survival of 59 days. Those patients receiving adjuvant chemotherapy or radiotherapy survived significantly longer than those receiving stenting alone (152.8 days vs. 71.8 days). There is no significant difference in complications or survival when using endoscopic or radiologic methods to deploy SEMs in patients with inoperable esophageal cancer. Mortality is low; however, the morbidity rate is significant. Patients receiving adjuvant chemotherapy or radiotherapy, in addition to stenting, survived significantly longer than those with a stent only.

Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53.

The p53 tumour suppressor is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation and proteasome-mediated degradation of p53. Many agents that induce p53 are inhibitors of transcription, suggesting that the p53 pathway can detect a signal(s) arising from transcriptional malfunction. Mdm2 associates with TAFII250, a component of the general transcription factor TFIID. Inactivation of TAFII250 in ts13 cells, which express a temperature-sensitive mutant of TAFII250, leads to the induction of p53 and cell cycle arrest. In the present study, we show that TAFII250 stimulates the ubiquitylation and degradation of p53 in a manner that is dependent upon Mdm2 and requires its acidic domain. Mechanistically, TAFII250 downregulates Mdm2 auto-ubiquitylation, leading to Mdm2 stabilization, and promotes p53-Mdm2 association through a recently defined second binding site in the acidic domain of Mdm2. These data provide a novel route through which TAFII250 can directly influence p53 levels and are consistent with the idea that the maintenance of p53 turnover is coupled to the integrity of RNA polymerase II transcription.

c-Abl phosphorylates Hdm2 at tyrosine 276 in response to DNA damage and regulates interaction with ARF.

The p53 tumour-suppressor protein is tightly regulated through its association with the Hdm2 E3 ligase. Activation of p53 by DNA strand breaks is orchestrated by the ataxia-telangiectasia mutated (ATM) protein kinase and involves interruption of Hdm2-mediated p53 degradation. As part of this mechanism ATM itself, and the ATM-activated protein tyrosine kinase, c-Abl, inhibit Hdm2 function through phosphorylation of serine 395 and tyrosine 394 (Y394), respectively. In the present study, we have identified a novel target of c-Abl in the Hdm2 protein, tyrosine 276 (Y276). We show that c-Abl phosphorylates this residue in vitro and confirm that Y394 is a target of c-Abl. We also show that Y276 is phosphorylated in a c-Abl-dependent manner in cultured cells and provide evidence that Y276 is phosphorylated in response to DNA damage coincident with the activation of c-Abl. Finally, we show that Y276 phosphorylation stimulates interaction with ARF, leading to increased levels of nucleolar Hdm2 and decreased turnover of p53. These data establish Y276 as a physiological target of c-Abl that contributes functionally to the induction of p53.

Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells.

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.

Shifting brain inhibitory balance and connectivity of the prefrontal cortex of adults with autism spectrum disorder.

Currently, there are no effective pharmacologic treatments for the core symptoms of autism spectrum disorder (ASD). There is, nevertheless, potential for progress. For example, recent evidence suggests that the excitatory (E) glutamate and inhibitory (I) GABA systems may be altered in ASD. However, no prior studies of ASD have examined the 'responsivity' of the E-I system to pharmacologic challenge; or whether E-I modulation alters abnormalities in functional connectivity of brain regions implicated in the disorder. Therefore, we used magnetic resonance spectroscopy ([1H]MRS) to measure prefrontal E-I flux in response to the glutamate and GABA acting drug riluzole in adult men with and without ASD. We compared the change in prefrontal 'Inhibitory Index'-the GABA fraction within the pool of glutamate plus GABA metabolites-post riluzole challenge; and the impact of riluzole on differences in resting-state functional connectivity. Despite no baseline differences in E-I balance, there was a significant group difference in response to pharmacologic challenge. Riluzole increased the prefrontal cortex inhibitory index in ASD but decreased it in controls. There was also a significant group difference in prefrontal functional connectivity at baseline, which was abolished by riluzole within the ASD group. Our results also show, for we believe the first time in ASD, that E-I flux can be 'shifted' with a pharmacologic challenge, but that responsivity is significantly different from controls. Further, our initial evidence suggests that abnormalities in functional connectivity can be 'normalised' by targeting E-I, even in adults.

Assessment and Optimisation of Lung Cancer Patients for Treatment with Curative Intent.

Over the past decade the field of lung cancer management has seen many developments. Coupled with an ageing population and increasing rates of comorbid illness, the work-up for treatments with curative intent has become more complex and detailed. As well as improvements in imaging and staging techniques, developments in both surgery and radiotherapy may now allow patients who would previously have been considered unfit or not appropriate for treatment with curative intent to undergo radical therapies. This overview will highlight published studies relating to investigation and staging techniques, together with assessments of fitness, with the aim of helping clinicians to determine the most appropriate treatments for each patient. We also highlight areas where further research may be required.

Mechanisms of switching on p53: a role for covalent modification?

The p53 protein plays a pivotal role in activating and integrating adaptive cellular responses to a wide range of environmental stresses. Activation of p53 can occur by different molecular routes, depending on the nature of the activating signal. Central to the activation process, by whichever route, is the destabilization of the p53-MDM2 interaction. The molecular mechanisms which activate p53 involve elements of post-translational modification, protein stabilization and protein-protein interaction. Two central themes are emerging from recent work in this area. The first is that there are common events in the p53 activation process among different activating pathways. The second is that activation involves not just a single molecular event such as disruption of the p53-MDM2 interaction, but a series of sequential events the nature of which is governed by the type of activating stimulus. This review summarizes our current knowledge of the p53 activation process in response to two stimuli, DNA damage and activated oncogenes, and considers the contribution made by multisite phosphorylation in determining the nature of the p53 response.

p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage.

The p53 tumour suppressor protein plays a central role in the cellular defence against agents which cause genetic damage. The activity of p53 is regulated at different levels and is subject to multi-site phosphorylation by a variety of different protein kinases. In this paper we have characterised p53 N-terminus-targeted protein kinase (p53NK) activities, present in a range of cell lines, following fractionation of cellular lysates by ion exchange chromatography on HiTrap Q and Mono Q resins. Three peaks of p53NK activity were observed following fractionation of HeLa cell lysates; these activities were each able to catalyse phosphorylation of up to three residues (serines 4, 6 and 9 in murine p53) within the N-terminus of the p53 protein. Similarly, multiple p53NK activities were detected in the MethAp53(ts) cell line (which expresses the valine 135 temperature-sensitive p53 protein). Strikingly, when these cells were shifted from 38 degrees C (the non-permissive temperature) to 28 degrees C, at which the p53 adopts a wild type conformation, a fivefold stimulation of kinase activity was detected. Moreover, when the DNA damage-inducing drugs etoposide or camptothecin were added to the cells, a further stimulation of kinase activity was observed following growth at 28 degrees C, but not 38 degrees C. These data are consistent with a regulatory model in which p53 is sensitive to stress or DNA damage through phosphorylation at its N-terminus.

Phosphorylation of murine p53, but not human p53, by MAP kinase in vitro and in cultured cells highlights species-dependent variation in post-translational modification.

The p53 tumour suppressor protein is tightly regulated by protein-protein association, protein turnover and a variety of post-translational modifications. Multisite phosphorylation plays a major role in activating and in finely tuning p53 function. The proline rich domain of murine p53 is a substrate for phosphorylation, in vitro and in cultured cells, by the p42ERK2 and p44ERK1 mitogen-activated protein (MAP) kinases. However, to date there have been no reports of attempts to determine whether p53 from any other species is a substrate for MAP kinase. In this paper we confirm that murine p53 is targeted by recombinant MAP kinase and by MAP kinases in extracts of both murine and human cells. In contrast, human p53 is not a substrate for recombinant MAP kinase nor are there any detectable levels of protein kinase activity in stimulated human cell extracts which phosphorylate the proline rich domain of human p53 in vitro. Finally, although stimulation of murine fibroblasts with o-tetradecanolylphorbol 13-acetate (TPA), an indirect activator of the MAP kinase pathway, leads to site-specific phosphorylation of murine p53, similar treatment of human fibroblasts and epithelial cells showed no significant changes in the phosphorylation pattern. These data are consistent with accumulating evidence that significant species-dependent differences exist in the post-translational modification of p53.

Phosphorylation of the p53 tumour-suppressor protein at three N-terminal sites by a novel casein kinase I-like enzyme.

Wild-type mouse p53, expressed in Escherichia coli, was phosphorylated by highly purified casein kinase I (CKI) from rabbit muscle. The major site of phosphorylation in the p53 was identified as serine 6, which is known to be phosphorylated in vivo. Serines 4 and 9 were also phosphorylated. To determine whether CKI is likely to be a physiological p53 kinase, SV3T3 cell lysates were fractionated on a Mono Q column and assayed for p53 kinase and casein kinase activities. Four p53 kinase activities were detected, one of which co-purified with CKI activity. This p53 kinase (designated PK270) further co-purified with CKI on sucrose gradients and had a native molecular weight, like CKI, in the range of 35,000-45,000. However, PK270 was separated from the bulk of CKI activity on a phosvitin-Sepharose affinity column, and was therefore likely to be a CKI-related kinase. In support of these conclusions, phosphorylation of p53, by both CKI and PK270, was inhibited by a peptide corresponding to a consensus CKI target sequence, but not by a non-specific peptide. Moreover, phosphopeptide analyses of p53 phosphorylated by CKI or by PK270 gave similar results, indicating that these two kinases phosphorylate the same sites in p53.

The conformational change of a murine temperature-sensitive p53 protein is independent of a change in phosphorylation status.

Wild-type and mutant p53 proteins exhibit opposing activities in respectively suppressing and promoting tumour development. In a rat embryo fibroblast cell line transformed with a murine temperature-sensitive p53 gene, p53 functions as a oncogene at 37 degrees C and as a tumour suppressor at 32 degrees C [Michalovitz, D., Halevy, O. & Oren, M. (1990). Cell, 62, 671-680]. We have used this cell line to investigate whether this temperature-dependent switching of function involves changes in the phosphorylation of p53 protein. Monoclonal antibodies PAb246 and PAb240 were used to immunoprecipitate metabolically 32P-labelled p53 protein in the 'wild-type' or mutant conformation from cells grown at 32 degrees C or 37 degrees C. Tryptic phosphopeptide maps were prepared from the isolated 'wild-type' and mutant p53 proteins. At 32 degrees C and 37 degrees C phosphopeptide maps of the 'wild-type' and mutant protein were identical. This demonstrates that the temperature-dependent conformation change, and associated functional change, in the p53 protein does not involve a change in the state of phosphorylation.

Murine p53 is phosphorylated within the PAb421 epitope by protein kinase C in vitro, but not in vivo, even after stimulation with the phorbol ester o-tetradecanoylphorbol 13-acetate.

The p53 tumour suppressor protein is thought to play a major role in the defence of the cell against agents which damage DNA. p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro. In this report, we have examined the phosphorylation of murine p53 by protein kinase C (PKC). Phosphopeptide mapping, phosphoamino acid analysis and radiosequence analysis of p53 phosphorylated by PKC in vitro indicated that serine 370 and threonine 377 were the major targets for phosphorylation and suggested that serine 372 and threonines 365 and 371 were minor phosphorylation sites. Site-directed mutagenesis confirmed that residues 370-372, all of which lie within the epitope for monoclonal antibody PAb421, were phosphorylated in vitro. The p53 from 32P-labelled SV3T3 cells showed a phosphopeptide pattern which includes peptides with mobilities similar to those arising from phosphorylation of residues 370-372 by PKC in vitro. Only two of these in vivo-labelled phosphopeptides co-migrated in two dimensions with peptides labelled in vitro within the PAb421 epitope and their phosphorylation was not stimulated by the addition of the PKC activator o-tetradecanoylphorbol 13-acetate (TPA) to the cells, even though this treatment led to a fourfold stimulation of p53 phosphorylation by MAP kinase. Moreover, when the p53 proteins containing mutations at residues 370-372 were expressed in COS cells, there was no loss of any of the in vivo phosphopeptides, indicating that phosphorylation within the PAb42I epitope was undetectable in the cell. These data suggest that p53 and PKC may not interact in vivo. The two-dimensional migration pattern of the novel group of peptides is consistent with phosphorylation of previously uncharacterised sites within the central DNA binding region of p53.

IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects.

The p53-targeted kinases casein kinase 1delta (CK1delta) and casein kinase 1epsilon (CK1epsilon) have been proposed to be involved in regulating DNA repair and chromosomal segregation. Recently, we showed that CK1delta localizes to the spindle apparatus and the centrosomes in cells with mitotic failure caused by DNA-damage prior to mitotic entry. We provide here evidence that 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261), a novel inhibitor of CK1delta and CK1epsilon, triggers the mitotic checkpoint control. At low micromolar concentrations IC261 inhibits cytokinesis causing a transient mitotic arrest. Cells containing active p53 arrest in the postmitotic G1 phase by blockage of entry into the S phase. Cells with non-functional p53 undergo postmitotic replication developing an 8N DNA content. The increase of DNA content is accompanied by a high amount of micronucleated and apoptotic cells. Immunfluorescence images show that at low concentrations IC261 leads to centrosome amplification causing multipolar mitosis. Our data are consistent with a role for CK1delta and CK1epsilon isoforms in regulating key aspects of cell division, possibly through the regulation of centrosome or spindle function during mitosis.

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs.

The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.

Pyruvate metabolism in Campylobacter spp.

The metabolism of pyruvate by Campylobacter spp. was investigated employing one- and two-dimensional 1H, 13C and 31P nuclear magnetic resonance spectroscopy. Metabolically competent cells incubated aerobically with pyruvate yielded acetate, acetolactate, alanine, formate, lactate, and succinate. The production of acetolactate, alanine and lactate indicated the presence of acetohydroxy acid synthase, alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton to the Kreb's cycle, and the observation of pyruvate dehydrogenase activities in bacterial lysates supported this interpretation. Generation of pyruvate from L-serine in incubations with intact cells and lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was also formed in cell suspensions and lysates from phosphoenol pyruvate. The existence of anaplerotic sequences involving phosphoenol pyruvate carboxykinase and a malic enzyme were established in bacterial lysates. The activities of enzymes involved in the biosynthesis of isoleucine and valine were measured. Addition of pyruvate to different solid culture media inhibited bacterial growth, and the inhibition was attributed to the accumulation of acetate and formate. The variety of products formed using pyruvate as the sole substrate and the existence of anaplerotic sequences and anabolic pathways which employ pyruvate, showed the important role of this metabolite in the energy and biosynthesis metabolism of Campylobacter spp.

Multisite phosphorylation and the integration of stress signals at p53.

The p53 tumour suppressor protein is a potent transcription factor that plays a major role in the defence against tumour development. p53 exists in a latent form that can be activated by a range of stresses including DNA damage, hypoxia, cytokines, metabolic changes, viral infection, and activated oncogenes. Activation of p53 can lead to cellular growth arrest prior to entry into either S phase or mitosis or can trigger cell death through apoptosis. The modification of p53 by multisite phosphorylation provides a potential link between stress signalling and the regulation of p53 activity, and there is now striking evidence that agents that activate p53 can lead to selective changes in its phosphorylation status. Topologically, the phosphorylation sites in p53 fall into two discrete functional domains. Four phosphorylation events take place within the N-terminal 83 amino acids containing the transactivation domain and a region involved in transcription-independent growth suppression. At least three of these modifications occur in response to agents that cause cellular stress such as DNA damage. At the C-terminus, there are three phosphorylation events, each of which can independently regulate the specific DNA-binding function of p53, suggesting convergent control by different signalling pathways. The multiplicity of these covalent modifications and their responsiveness to a wide range of signals suggest that p53 activity is tightly and coordinately controlled in response to stresses and changes in the cellular environment.