DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B.

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53-/- tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

Surgical factors in the prevention of infection following major lower limb amputation.

OBJECTIVES: Infection following major lower limb amputation is common but surgical influences on the rates of infection are not known. We aim to assess the influence of peri-operative surgical factors on outcome. DESIGN AND METHODS: Review of a prospective database included all patients undergoing a major lower limb amputation from March 2008 to July 2010. Infection was classified using Centre for Disease Control criteria and multivariate analysis performed to identify significant risk factors. RESULTS: 127 patients, median age 78 yrs (31-98) were included. 34.6% of patients developed a wound infection following surgery; 47.7% of which were classed as superficial incisional surgical site infections, with 52.3% being deep incisional surgical site infections. There was a higher infection rate in below knee than above knee amputations (p < 0.001). There was no relationship between the grade of the operating surgeon (p = 0.829), peri-operative antibiotics (p = 0.933), length of operation (p = 0.651), use of nerve catheter (0.267) and the post-operative presence of infection. There was a higher rate of infection with the use of suction drains (p < 0.05). The use of skin clips rather than sutures was associated with an increased rate of infection (p < 0.05). There was an increased need for revision surgery with the use of skin clips, although this was not significant (p = 0.07). CONCLUSIONS: Skin clips and surgical drains adversely influence the risk of infection in major limb amputation and their use should be avoided.

Prevention of infection in arterial reconstruction.

BACKGROUND: Arterial reconstructions with prosthetic graft materials or vein are susceptible to infection with a resultant high patient mortality and risk of limb loss. To reduce the risk of infection effective perioperative measures are essential. OBJECTIVES: To determine the effectiveness of perioperative strategies to prevent infection in patients undergoing peripheral arterial reconstruction. SEARCH STRATEGY: We searched the Cochrane Peripheral Vascular Diseases Group trials register (last searched May 2006) and the Cochrane Central Register of Controlled Trials (CENTRAL) (last searched Issue 2, 2006), and reference lists of relevant articles. SELECTION CRITERIA: All randomised controlled trials (RCTs) evaluating measures intended to reduce or prevent infection in arterial surgery. DATA COLLECTION AND ANALYSIS: AS and PSE independently selected and assessed the quality of included trials. Relative risk was used as a measure of effect for each dichotomous outcome. MAIN RESULTS: Thirty-five RCTs were included. Of these, 23 were trials of prophylactic systemic antibiotics, three of rifampicin-bonded grafts, three of preoperative skin antisepsis, two of suction wound drainage, two of minimally invasive in situ bypass techniques, and individual trials of intraoperative glove change and wound closure techniques. Wound infection or early graft infection outcomes were recorded in all trials. Only two trials, both of rifampicin bonding, followed up graft infection outcomes to two years. Trials of antibiotics versus placebo were of highest quality with six double-blind studies of the ten included. Prophylactic systemic antibiotics reduced the risk of wound infection (Relative Risk (RR) 0.25, 95% Confidence Interval (CI) 0.17 to 0.38) and early graft infection in a fixed-effect model (RR 0.31, 95% CI 0.11 to 0.85, P = 0.02). Antibiotic prophylaxis for greater than 24 hours appears to be of no added benefit (RR 1.28, 95% CI 0.82 to 1.98). There was no evidence that prophylactic rifampicin bonding to dacron grafts reduced graft infection at either one month (RR 0.63, 95% CI 0.27 to 1.49) or two years (RR 1.05, 95% CI 0.46 to 2.40). There was no evidence of a beneficial or detrimental effect on rates of wound infection with suction groin-wound drainage (RR 0.96 95% CI 0.50 to 1.86) or of any benefit from a preoperative bathing or shower regimen with antiseptic agents over unmedicated bathing (RR 0.97, 95% CI 0.70 to 1.36). AUTHORS' CONCLUSIONS: There is clear evidence of the benefits of prophylactic broad spectrum antibiotics. Many other interventions intended to reduce the risk of infection in arterial reconstruction lack evidence of effectiveness.

Reduction of insulin-stimulated glucose uptake in L6 myotubes by the protein kinase inhibitor SB203580 is independent of p38MAPK activity.

Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.

Tourniquets in arterial bypass surgery.

BACKGROUND: successful infra-popliteal bypass depends on precise, atraumatic technique in performing the distal anastomosis. The use of a tourniquet facilitates the distal anastomosis, reducing dissection, avoiding traumatising clamping of the vessels and providing an "uncluttered" operating field. Despite these advantages the technique is under-used. OBJECTIVES: to review the use of tourniquets in arterial reconstruction, with particular reference to safety issues and complications. DESIGN, METHODS AND MATERIALS: a Medline search was performed (last search Feb. 2000), and keywords from relevant papers were used to perform subsequent searches. References were reviewed from each relevant paper. RESULTS: no randomised controlled trials were found. The review details reported use of tourniquets in arterial reconstruction, including techniques, outcomes and potential complications. CONCLUSION: the use of a tourniquet is a safe and effective technique to facilitate arterial reconstruction.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-gamma uses a different signaling pathway.

STAT1 is an essential transcription factor for macrophage activation by IFN-gamma and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-alpha occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-gamma-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-gamma-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-alpha caused activation of p38 MAPK whereas IFN-gamma did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-alpha production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKalpha and beta but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-gamma-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Intra-abdominal bleeding caused by spontaneous rupture of an accessory spleen: the CT findings.

Accessory spleens are common. Their clinical importance lies in the need to include their removal when performing a splenectomy for primary haematological disorders, or as the source of 'preservable' splenic tissue in cases of ruptured primary spleen. Rupture of a normal spleen almost always occurs because of trauma, spontaneous rupture is rare. In pathological spleens, however, 'spontaneous' rupture is more widely reported, although it is argued that minor trauma is often still responsible in these cases. We report a case of spontaneous isolated rupture of a histologically normal accessory spleen and show the CT findings.

Paradoxical activation of Raf by a novel Raf inhibitor.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.

Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580.

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from 'non-specific' effects. Four events thought to occur upon activation of stress-activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen- and stress-activated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.

Acute non-traumatic arm ischaemia.

BACKGROUND: Much attention has been paid to the management of acute leg ischaemia. Acute arm ischaemia is perceived as less of a problem because the risk of limb loss is lower. After conservative treatment up to half the patients have late symptoms, such as forearm claudication. METHODS: This study was a review of all published English language data on acute arm ischaemia. The entire Medline database was searched and other references were derived from the material perused. There were no randomized or controlled studies. RESULTS: The incidence of acute arm ischaemia is one-fifth that of acute leg ischaemia. Patients with arm ischaemia tended to be older with a mean age of 74 years compared with 70 years for acute leg ischaemia. Since the development of the embolectomy catheter, embolectomy can be performed in most patients under local anaesthetic. Collected outcome included successful restoration of the circulation in 65-94 per cent of patients and amputation in 0-18 per cent. The mortality rate ranged from 0 to 19 per cent, despite the use of local anaesthesia, mostly from associated cardiac disease. Management by a vascular specialist may be beneficial, particularly in complex cases. CONCLUSION: An active approach to the management of acute arm ischaemia is safe and effective and reduces the risk of late disabling symptoms.

Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution.

BACKGROUND: Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38 beta 2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. RESULTS: The SAP kinases SAPK1 gamma/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38 beta 2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGF beta receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGF beta receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2, is inhibited by SB 203580. CONCLUSIONS: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38 beta 2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.

Regulation of System A amino acid transport in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress.

In this study we have investigated the effects of insulin, chemical and hyperthermic stresses upon the activity of the System A amino acid transporter in L6 rat muscle cells. Uptake of alpha-methyl-aminoisobutyric acid (Me-AIB), a non-metabolisable System A substrate, was increased by between 50% and 80% when muscle cells were exposed to a maximally effective concentration of insulin (100 nM), sodium arsenite (ARS, 0.5 mM) or a 42 degrees C heat shock (HS). The observed activation in System A in response to all three stimuli was maximal within 20 min and in the case of insulin and ARS primarily involved an increase in the Vmax of System A transport. In contrast, HS induced significant increases in both Vmax and Km of System A transport suggesting that hyperthermic stress may activate System A by a mechanism distinct from that mediating the effects of insulin and ARS. The hormonal stimulation of System A was blocked by the phosphoinositide 3-kinase (PI3k) inhibitor, wortmannin, but not by rapamycin or PD 98059 which respectively inhibit the mTOR and classical MAP kinase pathways. Exposure of L6 cells to ARS, but not HS, caused a 4.7-fold stimulation in MAPKAP-K2 activity that was blocked by SB 203580, a specific inhibitor of the stress activated protein kinase SAPK2/p38. However, neither SB 203580, rapamycin nor wortmannin were able to suppress the ARS- or HS-induced stimulation in System A transport. In summary, our results demonstrate that activity of the System A transporter can be rapidly upregulated in response to hormonal and stress stimuli through changes in the transport kinetics of the System A carrier. Our data show that whilst the hormonal response is PI3k dependent, the signalling mechanisms which instigate changes in System A activity in response to chemical or hyperthermic stress do not appear to involve PI3k or components of the mTOR, p42/p44 MAP kinase or SAPK2/p38 signalling pathways.

Inhibition of bovine nasal cartilage degradation by selective matrix metalloproteinase inhibitors.

N-terminal analysis of aggrecan fragments lost from bovine nasal cartilage cultured in the presence of recombinant human interleukin 1alpha revealed a predominant ARGSVIL sequence with an additional ADLEX sequence. Production of the ARGSVIL-containing fragments has been attributed to the action of a putative proteinase, aggrecanase. The minor sequence (ADLEX) corresponds to a new reported cleavage product; comparison of this sequence with the available partial sequence of bovine aggrecan indicates that this is the product of a cleavage occurring towards the C-terminus of the protein. Matrix metalloproteinase (MMP) inhibitors inhibited aggrecan loss from bovine nasal explants incubated in the presence of recombinant human interleukin 1alpha. A strong correlation between inhibition of aggrecan metabolism and inhibition of stromelysin 1 (MMP 3) (r=0.93) suggests a role for stromelysin or a stromelysin-like enzyme in cartilage aggrecan metabolism. However, the compounds were approx. 1/1000 as potent in inhibiting aggrecan loss from the cartilage explants as they were in inhibiting stromelysin. There was little or no correlation between inhibition of aggrecan metabolism and inhibition of gelatinase B (MMP 9) or inhibition of collagenase 1 (MMP 1). Studies with collagenase inhibitors with a range of potencies showed a correlation between inhibition of collagenase activity and inhibition of collagen degradation in the cartilage explant assay. This indicates that in interleukin 1alpha-driven bovine nasal cartilage destruction, stromelysin (or a closely related enzyme) is involved in aggrecan metabolism, whereas collagenase is principally responsible for collagen degradation.