Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Porcine CD38 exhibits prominent secondary NAD+ cyclase activity

Cyclic ADP‐ribose (cADPR) mobilizes intracellular Ca²⁺ stores and activates Ca²⁺ influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD⁺ by the multifunctional CD38/ADP‐ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD⁺ can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD⁺ to form linear ADPR while Aplysia ADP‐ribosyl cyclase prefers cyclizing NAD⁺ to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD⁺ cyclase activity producing cADPR. We also determined the X‐ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD⁺ reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.     

Novel Functions of the Phosphatase SHP2 in the DNA Replication and Damage Checkpoints

Replication stress- and DNA damage-induced cell cycle checkpoints are critical for maintaining genome stability. To identify protein phosphatases involved in the activation and maintenance of the checkpoints, we have carried out RNA interference-based screens with a human phosphatome shRNA library. Several phosphatases, including SHP2 (also called PTPN11) were found to be required for cell survival upon hydroxyurea-induced replicative stress in HeLa cells. More detailed studies revealed that SHP2 was also important for the maintenance of the checkpoint after DNA damage induced by cisplatin or ionizing radiation in HeLa cells. Furthermore, SHP2 was activated after replicative stress and DNA damage. Although depletion of SHP2 resulted in a delay in cyclin E accumulation and an extension of G₁ phase, these cell cycle impairments were not responsible for the increase in apoptosis after DNA damage. Depletion of SHP2 impaired CHK1 activation, checkpoint-mediated cell cycle arrest, and DNA repair. These effects could be rescued with a shRNA-resistant SHP2. These results underscore the importance of protein phosphatases in checkpoint control and revealed a novel link between SHP2 and cell cycle checkpoints.     

Trigeminal Neurosensory Deficit and Patient Reported Outcome Measures: The Effect on Life Satisfaction and Depression Symptoms

Objectives

To investigate the effect of persistent neurosensory disturbance of the lingual nerve (LN) or inferior alveolar nerve (IAN) on life satisfaction and depression symptoms.

Methods

This study recruited patients with persistent LN or IAN deficit as a consequence of lower third molar surgery for 12 months or more to form the study group. A control group was formed by matching age and gender of recruited subjects in the study group with patients without the neurosensory complications. Life satisfaction was assessed with Satisfaction With Life Scale (SWLS) and depression symptoms were assessed with 20-item Center for Epidemiological Studies Depression scale (CESD-20).

Results

Fourty-eight participants (24 cases and 24 controls) were recruited. Patients with LN or IAN deficits after lower third molar surgery were less satisfied with their lives when compared to the control group (p<0.001). They were presenting with more depression symptoms (p = 0.001). 45.8% of the study group subjects had a CESD-20 score of 16 or above. Older patients presented with more depression symptoms among the subjects with neurosensory disturbance after lower third molar surgery (p = 0.02).

Conclusions

Individuals with permanent trigeminal neurosensory deficit after lower third molar surgery have worse life satisfaction and more depressive symptoms when compared to those who did not suffer from the surgical complication.     

Development and Validation of the Chinese Attitudes to Starting Insulin Questionnaire (Ch-ASIQ) for Primary Care Patients with Type 2 Diabetes

Objectives

To develop and evaluate the psychometric properties of a Chinese questionnaire which assesses the barriers and enablers to commencing insulin in primary care patients with poorly controlled Type 2 diabetes.

Research Design and Method

Questionnaire items were identified using literature review. Content validation was performed and items were further refined using an expert panel. Following translation, back translation and cognitive debriefing, the translated Chinese questionnaire was piloted on target patients. Exploratory factor analysis and item-scale correlations were performed to test the construct validity of the subscales and items. Internal reliability was tested by Cronbach’s alpha.

Results

Twenty-seven identified items underwent content validation, translation and cognitive debriefing. The translated questionnaire was piloted on 303 insulin naïve (never taken insulin) Type 2 diabetes patients recruited from 10 government-funded primary care clinics across Hong Kong. Sufficient variability in the dataset for factor analysis was confirmed by Bartlett’s Test of Sphericity (P<0.001). Using exploratory factor analysis with varimax rotation, 10 factors were generated onto which 26 items loaded with loading scores > 0.4 and Eigenvalues >1. Total variance for the 10 factors was 66.22%. Kaiser-Meyer-Olkin measure was 0.725. Cronbach’s alpha coefficients for the first four factors were ≥0.6 identifying four sub-scales to which 13 items correlated. Remaining sub-scales and items with poor internal reliability were deleted. The final 13-item instrument had a four scale structure addressing: ‘Self-image and stigmatization’; ‘Factors promoting self-efficacy; ‘Fear of pain or needles’; and ‘Time and family support’.

Conclusion

The Chinese Attitudes to Starting Insulin Questionnaire (Ch-ASIQ) appears to be a reliable and valid measure for assessing barriers to starting insulin. This short instrument is easy to administer and may be used by healthcare providers and researchers as an assessment tool for Chinese diabetic primary care patients, including the elderly, who are unwilling to start insulin.     

Nebulized perflubron and carbon dioxide rapidly dilate constricted airways in an ovine model of allergic asthma

Background

The low toxicity of perfluorocarbons (PFCs), their high affinity for respiratory gases and their compatibility with lung surfactant have made them useful candidates for treating respiratory diseases such as adult respiratory distress syndrome. We report results for treating acute allergic and non-allergic bronchoconstriction in sheep using S-1226 (a gas mixture containing carbon dioxide and small volumes of nebulized perflubron). The carbon dioxide, which is highly soluble in perflubron, was used to relax airway smooth muscle.

Methods

Sheep previously sensitized to house dust mite (HDM) were challenged with HDM aerosols to induce early asthmatic responses. At the maximal responses (characterised by an increase in lung resistance), the sheep were either not treated or treated with one of the following; nebulized S-1226 (perflubron + 12% CO₂), nebulized perflubron + medical air, 12% CO₂, salbutamol or medical air. Lung resistance was monitored for up to 20 minutes after cessation of treatment.In additional naïve sheep, a segmental bronchus was pre-contracted with methacholine (MCh) and treated with nebulized S-1226 administered via a bronchoscope catheter. Subsequent bronchodilatation was monitored by real time digital video recording.

Results

Treatment with S-1226 for 2 minutes following HDM challenge resulted in a more rapid, more profound and more prolonged decline in lung resistance compared with the other treatment interventions. Video bronchoscopy showed an immediate and complete (within 5 seconds) re-opening of MCh-constricted airways following treatment with S-1226.

Conclusions

S-1226 is a potent and rapid formulation for re-opening constricted airways. Its mechanism(s) of action are unknown. The formulation has potential as a rescue treatment for acute severe asthma.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0098-x) contains supplementary material, which is available to authorized users.     

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to N^G-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOS^Ser1177) and Akt^Ser473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state.