Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1.     

Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.     

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Identification by families of pediatric adverse events and near misses overlooked by health care providers

Background:

Identifying adverse events and near misses is essential to improving safety in the health care system. Patients are capable of reliably identifying and reporting adverse events. The effect of a patient safety reporting system used by families of pediatric inpatients on reporting of adverse events by health care providers has not previously been investigated.

Methods:

Between Nov. 1, 2008, and Nov. 30, 2009, families of children discharged from a single ward of British Columbia’s Children’s Hospital were asked to respond to a questionnaire about adverse events and near misses during the hospital stay. Rates of reporting by health care providers for this period were compared with rates for the previous year. Family reports for specific incidents were matched with reports by health care providers to determine overlap.

Results:

A total of 544 familes responded to the questionnaire. The estimated absolute increase in reports by health care providers per 100 admissions was 0.5% (95% confidence interval −1.8% to 2.7%). A total of 321 events were identified in 201 of the 544 family reports. Of these, 153 (48%) were determined to represent legitimate patient safety concerns. Only 8 (2.5%) of the adverse events reported by families were also reported by health care providers.

Interpretation:

The introduction of a family-based system for reporting adverse events involving pediatric inpatients, administered at the time of discharge, did not change rates of reporting of adverse events and near misses by health care providers. Most reports submitted by families were not duplicated in the reporting system for health care providers, which suggests that families and staff members view safety-related events differently. However, almost half of the family reports represented legitimate patient safety concerns. Families appeared capable of providing valuable information for improving the safety of pediatric inpatients.It has been estimated that adverse events occur in about 1% of children treated in hospital and that, on average, 60% of these events are preventable.¹ To increase institutional awareness of adverse events, hospitals have implemented systems to encourage health care providers to report adverse events.² The reporting of adverse events can be improved by making electronic systems for reporting readily accessible³ and by ensuring a “just culture,” which includes nonpunitive reporting policies.⁴ However, adverse events reported by health care providers account for only a small fraction of total adverse events as determined by chart review.⁵ Time pressures to treat patients, fear of punishment, lack of belief in the benefit of reporting and differing opinions of what defines a reportable event contribute to low reporting rates.⁶ However, patients and their families are readily available, keen and motivated observers who may not be subject to these reporting barriers. Family members are capable of observing and reporting adverse events in a variety of clinical settings.⁷ It is known that the interpretation of safety events and the threshold for reporting differ among health care disciplines and individual health care providers.⁶ However, it is not clear how families of pediatric patients interpret safety-related events or what their threshold would be for reporting events.The purpose of this study was to test whether the introduction of an adverse event reporting system for use by families of pediatric patients at the time of discharge from a surgical ward would significantly change the rate of reporting of adverse events by health care providers. We also evaluated the types of events that families reported, the relevance of these events with respect to patient safety, families’ desires for anonymous reporting and families’ assessments of institutional responses to reported events. We anticipated that health care providers’ reporting rates would rise with the introduction of the family reporting system, on the assumption that greater attention would be paid to reporting safety-related events on the ward. We also anticipated that families would provide useful information about safety-related events, at least some of the time. In particular, we thought that facilitating communication from the patient’s family directly to the study institution’s Quality, Safety and Outcome Improvement Department would allow more opportunities to improve safety through changes in practice.     

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.     

Mechanisms and consequences of TGF-? overexpression by podocytes in progressive podocyte disease

In patients with progressive podocyte disease, such as focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, upregulation of transforming growth factor-? (TGF-?) is observed in podocytes. Mechanical pressure or biomechanical strain in podocytopathies may cause overexpression of TGF-? and angiotensin II (Ang II). Oxidative stress induced by Ang II may activate the latent TGF-?, which then activates Smads and Ras/extracellular signal-regulated kinase (ERK) signaling pathways in podocytes. Enhanced TGF-? activity in podocytes may lead to thickening of the glomerular basement membrane (GBM) by overproduction of GBM proteins and impaired GBM degradation in podocyte disease. It may also lead to podocyte apoptosis and detachment from the GBM, and epithelial-mesenchymal transition (EMT) of podocytes, initiating the development of glomerulosclerosis. Furthermore, activated TGF-?/Smad signaling by podocytes may induce connective tissue growth factor and vascular endothelial growth factor overexpression, which could act as a paracrine effector mechanism on mesangial cells to stimulate mesangial matrix synthesis. In proliferative podocytopathies, such as cellular or collapsing FSGS, TGF-?-induced ERK activation may play a role in podocyte proliferation, possibly via TGF-?-induced EMT of podocytes. Collectively, these data bring new mechanistic insights into our understanding of the TGF-? overexpression by podocytes in progressive podocyte disease.     

Effects of heat acclimatisation on work tolerance and thermoregulation in trained tropical natives

This study aimed to investigate the effects of heat acclimatisation on thermoregulatory responses and work tolerance in trained individuals residing in the tropics. Eighteen male trained soldiers, who are native to a warm and humid climate, performed a total of four heat stress tests donning the Skeletal Battle Order (SBO, 20.5 kg) and Full Battle Order (FBO, 24.7 kg) before (PRE) and after (POST) a 10-day heat acclimatisation programme. The trials were conducted in an environmental chamber (dry bulb temperature: 32 °C, relative humidity: 70%, solar radiation: 400 W/m²). Excluding the data sets of which participants fully completed the heat stress tests (210 min) before and after heat acclimatisation, work tolerance was improved from 173±30 to 201±18 min (∼21%, p<0.05, n=9) following heat acclimatisation. Following heat acclimatisation, chest skin temperature during exercise was lowered in SBO (PRE=36.7±0.3 vs. POST=36.5±0.3 °C, p<0.01) and FBO (PRE=36.8±0.4 vs. POST=36.6±0.3 °C, p<0.01). Ratings of perceived exertion were decreased with SBO and FBO (PRE=11±2; POST=10±2; p<0.05) after heat acclimatisation. Heat acclimatisation had no effects on baseline body core temperature, heart rate and sweat rate across trials (p>0.05). A heat acclimatisation programme improves work tolerance with minimal effects on thermoregulation in trained tropical natives.     

JNK3 Perpetuates Metabolic Stress Induced by Aβ Peptides

Although Aβ peptides are causative agents in Alzheimer's disease (AD), the underlying mechanisms are still elusive. We report that Aβ42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aβ42 in a JNK3-dependent manner. Thus, JNK3 activation, which is?increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aβ42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aβ42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.