Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL. Subsequent studies revealed a reduction of the mitochondrial membrane potential and caspase-3 activation in these two cell lines upon treatment with proteasome inhibitors; however, caspase-3 activation occurred much earlier in Jurkat cells. Cell cycle analysis indicated a sub-G(1) apoptotic cell population in Jurkat cells and G(2)/M arrest in YT cells after they were treated by proteasome inhibitors. Moreover, pretreatment of YT cells by a caspase inhibitor followed by a proteasome inhibitor did not increase the percentage of G(2)/M phase cells. In addition, accumulation of p27 and IkappaB-alpha was detected only in Jurkat cells, but not YT cells. In summary, proteasome inhibitors may act differentially in cell cycle arrest and apoptosis of tumors of NK and T cell origin, and may have similar effects on normal NK and T cells.     

Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5'- and 3'-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5'- and 3'-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5'-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5'-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, approximately 50% of duplicated 3'-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.     

内皮祖细胞及其在组织工程中的应用

目前,组织工程化血管的构建和工程化组织器官的血管化因内皮种子细胞的扩增能力不足和生物活性不强而受到限制。内皮祖细胞(EPC)是内皮细胞的前体细胞。出生后,EPC主要存在于骨髓,可向外周血液缓慢释放,参与机体缺血组织的血管重建和损伤血管的重新内皮化。现对EPC的来源、分布、表型特征、动员、分化、归巢、分离、培养与鉴定等生物学特性和EPC在组织工程中的应用进行了全面的综述,并指出目前存在的问题和研究方向。     

Determination of compound aminopyrine phenacetin tablets by using artificial neural networks combined with principal components analysis

A method for simultaneous, nondestructive analysis of aminopyrine and phenacetin in compound aminopyrine phenacetin tablets with different concentrations has been developed by principal component artificial neural networks (PC-ANNs) on near-infrared (NIR) spectroscopy. In PC-ANN models, the spectral data were initially analyzed by principal component analysis. Then the scores of the principal components were chosen as input nodes for the input layer instead of the spectral data. The artificial neural network models using the spectral data as input nodes were also established and compared with the PC-ANN models. Four different preprocessing methods (first-derivative, second-derivative, standard normal variate (SNV), and multiplicative scatter correction) were applied to three sets of NIR spectra of compound aminopyrine phenacetin tablets. The PC-ANNs approach with SNV preprocessing spectra was found to provide the best results. The degree of approximation was performed as the selective criterion of the optimum network parameters.     

Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.     

Molecular characterization,tissue distribution and expression analysis of Interleukin-12 receptor β2 chain in sheep

Ovine β2 subunit of the interleukin (IL)-12 receptor (IL-12Rβ2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rβ2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rβ2. The ovine IL-12 Rβ2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rβ2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rβ2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rβ2 mRNA expression was detected at all the detected tissues with the exception of thymus.     

Complete Genome Sequence of a Duck Hepatitis A Virus Type 3 Identified in Eastern China

We report here the complete genome sequence of a novel duck hepatitis A virus type 3 (DHAV-3) isolated from a dead Cherry Valley duckling in eastern China. The whole genomic nucleotide sequence and polyprotein amino acid sequence of the virus had higher homology with those of Chinese DHAV-3 isolates, medium homology with those of Korean DHAV-3 isolates, and the lowest homology with those of Vietnamese isolate DN2. The result indicated that the genetic evolution of DHAV-3 isolates had obvious geographical features.     

Purification and partial characterization of glutathione S-transferases from three field populations of Panonychus citri (Acari: Tetranychidae)

Glutathione S-transferases (GSTs) play central roles in phase II detoxification of both xenobiotics (drugs, insecticides, and herbicides) and endogenous compounds in almost all living organisms. In this study, we successfully purified the GSTs from the citrus red mite, Panonychus citri, by affinity chromatography on Glutathione Sepharose 4B and compared the biochemical characterizations of the purified GSTs from three field populations [beibei (BB), wanzhou (WZ), and zhongxian (ZX)]. SDS–PAGE revealed that the molecular weight of GSTs from three populations consisted of two subunits of 27.3 and 26.1 kDa. The specific activity of the purified GSTs from the WZ and ZX populations was increased 1.5- and 3.8-fold, respectively, compared with the BB population. Accordingly, the pyridaben susceptibility of WZ and ZX populations was less compared with BB population. Kinetic analyses showed that the WZ and ZX populations had higher substrate specificity compared with the BB population based on the values of k _cat and k _cat /K _m to both reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). The in vitro inhibition studies of GSTs indicated that the I ₅₀ values of pyridaben from WZ and ZX populations of P. citri expressed 1.6- and 4.4-fold decreases, respectively, compared to the I ₅₀ value of pyridaben from the BB population. In conclusion, all evidence suggested that the purified GSTs may partially contribute to the susceptibility of acaricide pyridaben in field populations of P. citri.     

Laparoscope resection of ectopic corticosteroid-secreting adrenal adenoma

Tumors originating from ectopic adrenal tissue are relatively rare. In this article, we describe a case with Cushing's syndrome caused by an ectopic adrenal adenoma. A 38 year-old male patient presenting with cushingoid appearance for 2 years was diagnosed to have ACTH-independent Cushing's syndrome based on endocrinological evaluation. Mutiple radiological examinations detected bilateral adrenal atrophy. When the images were investigated in a more expanded scope, a 3.0×3.5×5.3?cm mass was detected in the anterior of left renal hilum and left renal vein. The mass was successfully resected with intraoperative endoscopy and pathological evaluation revealed an ectopic adrenal tumor. It is suggested that when the endocrinlogically confirmed adrenal neoplasm could not be well and definitely localized, the possibility of ectopic adrenal should be presumed and further radiography examinations should extend to the field where ectopic adrenal usually presents.