Cloning and biological activity of an anti-tumor peptide of Tumstatin

To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203 amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction, the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC). The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide has the potential to become a novel, potent anti-tumor agent. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报]     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

The side population in human lung cancer cell line NCI-H460 is enriched in stem-like cancer cells

Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate. Recent studies indicate that tumors contain a subset of stem-like cancer cells that possess certain stem cell properties. Herein, we used Hoechst 33342 dye efflux assay and flow cytometry to isolate and characterize the side population (SP) cells from human lung cancer cell line NCI-H460 (H460). We show that the H460 SP cells harbor stem-like cells as they can readily form anchorage-independent floating spheres, possess great proliferative potential, and exhibit enhanced tumorigenicity. Importantly, the H460 SP cells were able to self-renew both in vitro and in vivo. Finally, we show that the H460 SP cells preferentially express ABCG2 as well as SMO, a critical mediator of the Hedgehog (HH) signaling, which seems to play an important role in H460 lung cancer cells as its blockage using Cyclopamine greatly inhibits cell-cycle progression. Collectively, our results lend further support to the existence of lung cancer stem cells and also implicate HH signaling in regulating large-cell lung cancer (stem) cells.     

PCR-based generation of shRNA libraries from cDNAs

Background  

The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.     

Soil organic carbon changes as influenced by agricultural land use and management: a case study in Yanhuai Basin, Beijing, China

The effects of agricultural land use and management practices on soil organic carbon (SOC) are of great concern. In this study, SOC changes were investigated in sandy loam soils (Ustochrepts, USDA Soil Taxonomy) under orchard, vegetable, corn (Zea maize L.), and soybean (Glycine max L.) cultivation in northern China. The corn fields were further classified into three categories based on their inputs, i.e. high-input, mid-input, and low-input corn fields. In April 2005, a total of 197 soil samples were collected from 42 soil sites within 100 cm soil depth in Yanhuai Basin, Beijing, China. SOC contents were determined using rapid dichromate oxidation, and ANOVA statistical analysis was used to test the significances between land use and management practices at p<0.05. The results showed that: (1) the effects of land use and management practices on SOC primarily occurred within the topsoil (0–25 cm), and the SOC contents sharply decreased with the increase in soil depth. (2) SOC content and density values of orchard, vegetable, and high-input corn fields were higher than those of soybean, mid- and low-input corn fields.