Dynamic changes of microtubule and actin structures in CV-1 cells during electrofusion

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV-1 cells were fused by applying a radio-frequency electrical pulse. Structural changes of microtubules (MTs) and F-actin were monitored simultaneously by double-label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of F-actin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring-shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.     

Fine mapping of the MLK-3 gene within 11q13 and its exclusion as the MEN1 susceptibility gene

MLK-3 kinase is a widely expressed serine/ threonine kinase that bears multiple protein interaction domains and regulates signals mediated by the stress-responsive pathway. Thus, MLK-3 signaling affects numerous cellular processes, raising the possibility that MLK-3 might play a role in oncogenesis. In this report, we describe the fine mapping of the MLK-3 gene within the 11q13.1 chromosomal region. By integrating data from somatic cell hybrids and double color fluorescence in situ hybridization on metaphase chromosomes and DNA fibers, MLK-3 has been assigned approximately 1 Mb telomeric of PYGM, close to the D11S546 locus. Since the MEN1 susceptibility locus is also located within the 11q13.1 region, we have carried out Southern and Northern blot analyses, as well as protein truncation assays to establish whether abnormalities in MLK-3 lead to the development of this familial cancer syndrome. Our observations exclude MLK-3 as the MEN1 gene. Received: 25 September 1996 / Revised: 16 December 1996     

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Effect of Heterogeneous Investments on the Evolution of Cooperation in Spatial Public Goods Game

Understanding the emergence of cooperation in spatial public goods game remains a grand challenge across disciplines. In most previous studies, it is assumed that the investments of all the cooperators are identical, and often equal to 1. However, it is worth mentioning that players are diverse and heterogeneous when choosing actions in the rapidly developing modern society and researchers have shown more interest to the heterogeneity of players recently. For modeling the heterogeneous players without loss of generality, it is assumed in this work that the investment of a cooperator is a random variable with uniform distribution, the mean value of which is equal to 1. The results of extensive numerical simulations convincingly indicate that heterogeneous investments can promote cooperation. Specifically, a large value of the variance of the random variable can decrease the two critical values for the result of behavioral evolution effectively. Moreover, the larger the variance is, the better the promotion effect will be. In addition, this article has discussed the impact of heterogeneous investments when the coevolution of both strategy and investment is taken into account. Comparing the promotion effect of coevolution of strategy and investment with that of strategy imitation only, we can conclude that the coevolution of strategy and investment decreases the asymptotic fraction of cooperators by weakening the heterogeneity of investments, which further demonstrates that heterogeneous investments can promote cooperation in spatial public goods game.     

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.     

Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum

Applied Microbiology and Biotechnology - Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides...