eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

Effect of Bone Mineral Density on Rotator Cuff Tear: An Osteoporotic Rabbit Model

IntroductionAn increased bone mineral density (BMD) in the proximity to tendon insertion can improve rotator cuff repair and healing. However, how a decrease of BMD in the humeral head affects the biomechanical properties of the rotator cuff tendon is still unclear. Previous studies have demonstrated ovariectomy in animals to lead to osteoporosis and decreased BMD, and Teriparatide (PTH) administration to improve BMD and strength of bone. This study aimed to explore the correlation between humeral head BMD and infraspinatus (ISP) tendon insertion strength, and if an increase in bone quantity of the humeral head can improve the strength of the rotator cuff.ResultsSignificant differences were observed in the measured humeral head BMD of the Control and OVX-PTH groups compared to the OVX-Saline group (P = 0.0004 and P = 0.0024, respectively). No significant difference was found in failure stress among the three groups, but an expected trend with the control group and OVX-PTH group presenting higher failure strength compared to the OVX-Saline group. BMD at the humeral head showed a positive linear correlation with stress (r² = 0.54). Histology results showed the superiority in OVX-PTH group ISP enthesis compared to the OVX-Saline group.ConclusionBone loss of the humeral head leads to decreased tendon/bone insertion strength of the infraspinatus tendon enthesis. Teriparatide administration can increase bone density of the humeral head and may improve the mechanical properties of the infraspinatus tendon enthesis.     

A new genus of Coelotinae (Araneae,Agelenidae) from southern China

One new genus of the spider subfamily Coelotinae, Flexicoelotes gen. n., with five new species is described from southern China: Flexicoelotes huyunensis sp. n. (female), Flexicoelotes jiaohanyanensis sp. n. (male and female), Flexicoelotes jinlongyanensis sp. n. (male and female), Flexicoelotes pingzhaiensis sp. n. (female), Flexicoelotes xingwangensis sp. n. (male and female).     

Statistical Inference Methods for Two Crossing Survival Curves: A Comparison of Methods

A common problem that is encountered in medical applications is the overall homogeneity of survival distributions when two survival curves cross each other. A survey demonstrated that under this condition, which was an obvious violation of the assumption of proportional hazard rates, the log-rank test was still used in 70% of studies. Several statistical methods have been proposed to solve this problem. However, in many applications, it is difficult to specify the types of survival differences and choose an appropriate method prior to analysis. Thus, we conducted an extensive series of Monte Carlo simulations to investigate the power and type I error rate of these procedures under various patterns of crossing survival curves with different censoring rates and distribution parameters. Our objective was to evaluate the strengths and weaknesses of tests in different situations and for various censoring rates and to recommend an appropriate test that will not fail for a wide range of applications. Simulation studies demonstrated that adaptive Neyman’s smooth tests and the two-stage procedure offer higher power and greater stability than other methods when the survival distributions cross at early, middle or late times. Even for proportional hazards, both methods maintain acceptable power compared with the log-rank test. In terms of the type I error rate, Renyi and Cramér—von Mises tests are relatively conservative, whereas the statistics of the Lin-Xu test exhibit apparent inflation as the censoring rate increases. Other tests produce results close to the nominal 0.05 level. In conclusion, adaptive Neyman’s smooth tests and the two-stage procedure are found to be the most stable and feasible approaches for a variety of situations and censoring rates. Therefore, they are applicable to a wider spectrum of alternatives compared with other tests.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G_2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')₂ fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3–4 h of incubation at 37°C. The F(ab')₂ fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')₂ fragments for the diagnosis of cancer in patients.