Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.     

Age determines memory for face identity and expression

Background: The recognition of facial expressions is an important component of emotion processing which contributes to interactional behavior. One of the factors highly associated with potential decline of ability in behavioral tasks is age. Methods: We have investigated age‐related changes in facial identity and expression memory of healthy subjects in three age groups: young adults (20–40 years), elderly adults (60–80 years) and, for the first time in the literature, very old adults (over 80 years of age). Using a picture test, photographs of faces with happy or angry expressions were presented to study participants during the encoding task, and the memory for identity and emotional facial expression was investigated in a subsequent recognition task showing emotionally neutral faces. Half of the faces presented in the recognition task were initially shown in the encoding task. Results: Age interacted with the memory process: the ability to recognize both facial identity and emotional expression declined with advanced age. Happy facial expressions were better recognized in all age groups. Although there was a continuous overall decrease in recognition of both happy and angry expressions with advanced age, the effect favoring happy facial expressions was stable also in very old adults. Other factors such as gender or educational level did not affect the memory process for facial expressions. Conclusions: Our findings suggest that age is a significant determinant of memory for facial identity and emotional expression, and that, similar to younger adults, the recognition process of the elderly favors happy emotional facial expressions.     

When the standards aren't standard: Evidence-based medicine in the Russian context

This paper examines the uses of evidence-based medicine (EBM) in post-Soviet Russia through the use of semi-structured interviews. It asks what it means to talk about practicing evidence-based medicine in a setting where the context of practice presents considerable barriers to the implementation of EBM principles. Drawing on interviews with Russian physicians, medical students and users of the healthcare system, the paper argues that in post-Soviet Russia EBM serves as a strategic discourse for segments of the medical profession. With the collapse of the U.S.S.R. the healthcare system has been going through a period of crisis, and Russian physicians are finding that they have to redefine their professional identity with respect to the domestic and the international context and have to seek new sources for legitimating their professional position. The western origins of EBM endow this rhetoric with considerable power in the Russian context and render it a very useful tool in the project of redefinition.     

Factor Xa-activated whole blood clotting time (Xa-ACT) for bedside monitoring of dalteparin anticoagulation during haemodialysis.

BACKGROUND: Low molecular weight heparins (LMWH) like dalteparin are increasingly used for anticoagulation during haemodialysis (HD). The available laboratory tests for monitoring LMWH anticoagulation are time-consuming and expensive, and the suitability of the conventional activated clotting time (ACT) is controversial. A simple and cheap bedside test would be useful. METHODS: We studied the factor Xa-activated whole blood clotting time (Xa-ACT) in vitro and in vivo in nine patients undergoing chronic HD with i.v. dalteparin bolus anticoagulation and compared it with the conventional ACT. Plasma anti-factor Xa (antiXa) activity was determined with a chromogenic assay. Thrombin-antithrombin complexes were measured to detect coagulation activation. RESULTS: Xa-ACT and ACT were prolonged with rising dalteparin concentration. In vitro, both clotting times were strongly correlated with the antiXa levels (r = 0.94 and 0.89, respectively). Nevertheless, compared with the ACT, the Xa-ACT was considerably more sensitive to the LMWH in vitro (healthy blood: Xa-ACT 90 s/U vs ACT 26 s/U; uraemic blood: Xa-ACT 96 s/U vs ACT 31 s/U) as well as in vivo (Xa-ACT 81 s/U vs ACT 22 s/U) and reflected different intensities of anticoagulation. An initial dalteparin bolus of 80+/-11 U/kg body weight was able to prevent coagulation activation for up to 4 h of HD. CONCLUSION: For monitoring LMWH anticoagulation the Xa-ACT was superior to the conventional ACT in vitro as well as in vivo during HD. The Xa-ACT can be useful as a LMWH bedside test. The ACT was not sensitive enough to serve as a LMWH monitoring tool.     

A binding study of phospholipase A2 with lecithin,lysolecithin and their tetrahedral intermediates using molecular modeling

We used molecular modeling to examine the binding of 1, 2-dioctanoyl-sn-glycero-3-phosphocholine (a lecithin), 1-octanoyl-sn-glycero-3-phosphocholine (a lysolecithin) and their tetrahedral intermediates in the catalytic site of phospholipase A₂ (PLA₂). We performed energy minimization on each complex, computed the binding energy, determined the relative binding energy among the complexes and calculated the difference in inter- and intramolecular energies of the components in the complexes. We found that the calculated orientation of the sn-1 ester bond of lysolecithin in the active site is similar to that of the sn-2 ester bond in lecithin, thus permitting PLA₂ to hydrolyze lysolecithin using the same mechanism as it uses to hydrolyze lecithin. On the other hand, the binding of lecithin is energetically more favorable by 4.5 kcal/mol than the binding of lysolecithin to the enzyme, and the binding of the lecithin tetrahedral intermediate is also energetically more favorable by 19.7 kcal/mol than the binding of the lysolecithin tetrahedral intermediate to the enzyme, which explains why lecithin is a better substrate than lysolecithin in the catalytic site. These results indicate that the activation energy for the hydrolysis of lysolecithin is higher than that for lecithin, consistent with the observed slower rate for the hydrolysis of lysolecithin.     

Smoking delays chondrogenesis in a mouse model of closed tibial fracture healing.

Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model.     

Standardized molecular typing of Candida albicans strains

Summary. A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of Eco RI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.
Zusammenfassung. Zur Standardisierung des DNA-Fingerprinting von Candida albicans wurde eine Methode entwickelt, die auf der Southern Hybridisierung Eco RI-gespaltener chromosomaler DNA mit dem mittelrepetitiven DNA-Element CARE-2 und der darauffolgenden Rehybridisierung der Blots mit einem auch in den Proben enthaltenen molekularen Größenmarker beruht. Dies resultierte in einer äußerst präzisen Größen-bestimmung der hybridisierenden Fragmente, so daß alle stammspezifischen CARE-2-Hybridisierungsmuster exakt verglichen werden konnten, auch wenn die Isolate auf verschiedenen Gelen analysiert wurden. Die Methode erhöht die Genauigkeit der Bestimmung genetischer Verwandtschaftsbeziehungen in epidemiologischen Untersuchungen, in denen eine große Anzahl von Stämmen analysiert wird.     

云南罗平医院员工对医院的认同度分析

通过问卷调查和访谈分析,调查了云南罗平医院194名员工对医院的认同度,调查对象包括行政、临床和后勤人员,从医院管理模式、医院绩效考核、医院薪酬待遇等方面用描述统计方法分析了数据,显示了医院员工的认同度情况及管理中存在的问题,并分析了不同因素对医院员工的认同度影响,为进一步改善医院管理状况,提高管理水平提供了参考。