MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization.

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana. The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.

EVL (Ena/VASP-like) expression is up-regulated in human breast cancer and its relative expression level is correlated with clinical stages.

EVL belongs to the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family of proteins [Mena (mammalian Ena), VASP and EVL], which have a range of roles in regulating the actin cytoskeleton. Growing evidence suggests that Mena and VASP are involved in carcinogenesis, though little is known about the significance of EVL's function in cancer. This study examined the expression levels of EVL mRNA in paired breast cancer specimens using semi-quantitative and real-time RT-PCR. In a comparison between matched breast tumors and normal tissues, a significant increase in the level of EVL mRNA was found in 23 of the 35 (65.7%) tumors (P=0.032). Patients in the advanced stages more frequently exhibited an elevated EVL expression in tumor tissues. The EVL mRNA relative expression level significantly correlated with clinical stages (P=0.021). To begin elucidating the mechanism, we measured the ability of EVL to transform NIH3T3 cells and regulate the motility of MCF-7 cells in vitro by focus formation and modified Boyden chamber assays. The results indicated that overexpression of EVL was insufficient to transform NIH3T3 cells, although the motility of EVL transfected MCF-7 cells was markedly promoted. Collectively, EVL expression level was higher in breast tumors compared to normal tissues and its up-regulation was positively associated with the clinical stages of breast cancer. Additionally, EVL may be implicated in invasion and/or metastasis of human breast cancer.

Biphasic expression of RhoGDI2 in the progression of breast cancer and its negative relation with lymph node metastasis.

RhoGDI2 has been shown to be a metastasis-related gene in several cancers. In human breast cancer, little clinical study of RhoGDI2 has been reported. In this study, we investigated the expression level of RhoGDI2 by immunohistochemistry, as well as the correlation of RhoGDI2 with clinicopathological parameters in 71 breast cancer specimens. We also examined RhoGDI2 expression at mRNA and protein levels of four human breast cancer cell lines differing in in vivo metastasis. Along with the extent of mammary epithelia proliferation and carcinogenesis, a biphasic pattern of RhoGDI2 expression (increase and then decrease) was observed, which was also found in these examined cells. Furthermore, univariate and multivariate analysis revealed that reduced expression of RhoGDI2 in the most malignant epithelia was significantly associated with lymph node metastasis (P<0.01). Our results suggest that RhoGDI2 may be implicated in the progress of malignancy and act as a metastasis-related marker in breast cancer.

Functional characterization of a putative nitrate transporter gene promoter from rice.

Drought is one of the most significant abiotic stresses that influence plant growth and development. Expression analysis revealed that OsNRT1.3, a putative nitrate transporter gene in rice, was induced by drought. To confirm if the OsNRT1.3 promoter can respond to drought stress, a 2019 bp upstream sequence of OsNRT1.3 was cloned. Three OsNRT1.3 promoter fragments were generated by 5'-deletion, and fused to the beta-glucuronidase (GUS) gene. The chimeric genes were introduced into rice plants. NRT2019::GUS, NRT1196::GUS and NRT719::GUS showed similar expression patterns in seeds, roots, leaves and flowers in all transgenic rice, and GUS activity conferred by different OsNRT1.3 promoter fragments was significantly upregulated by drought stress, indicating that OsNRT1.3 promoter responds to drought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.

Isolation and characterization of OsGSTL1 promoter from rice.

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the beta-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2,155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2,155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1,224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5'-upstream region between -2,155 and -1,224 bp.

[Construction of the mutants of rice nonspecific lipid transfer protein and expression comparison in two kinds of thioredoxin fusion expression vectors].

Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site-directed mutagenesis. Sequence results showed that they were all mutated successfully. After trying various E. coli expression systems, thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110. cDNA sequences encoding wild type LTP110 and the mutants Y17A, P72L, R46A, D43A, C50A were cloned into two kinds of thioredoxin fusion expression vectors. The expression results were compared. In pTrxFus/GI724 expression system, wild type LTP110 and the mutants Y17A, P72L, R46A could be expressed at low level while D43A and C50A could not be expressed normally; in pET32a(+)/BL21 (DE3) trxB- expression system, wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system. LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid. Results indicated that the recombinant LTP110 fusion protein has lipid binding activity. This work provides good basis for the further study.

[Expression, purification and function of rice nonspecific lipid transfer protein].

Plant nonspecific lipid transfer protein(nsLTP) is a class of protein which has in vitro lipid transferring activity between biomembranes. In order to study the antimicrobial function of rice nonspecific lipid transfer protein, a gene LTP110 encoding rice nsLTP was cloned into ThioFusion expression vector pET32a (+) and expressed in host strain Bl21(DE3)trxB-. After induction by IPTG at 30 degrees C for 5 h, the fusion protein thio-LTP110 was in large amount produced. The expressed protein was purified by Ni2(+)-chelating Sepharose fast flow column, then digested by enterokinase. By passing through nickel affinity column again, the cleavage product, LTP110, was obtained. CD spectrum scanning from 185 nm to 250 nm showed that the recombinant protein LTP110 had similar secondary structure with the nsLTP purified from rice etiolated seedlings. Activity determination by fluorescent lipid P-96 showed that it had lipid binding activity. Microbial inhibition test results revealed that LTP110 deterred germination of the spores of rice pathogen P. oryzae, showing it might be involved in plant microbial resistance function. Therefore, it has the potential to be used in plant transgene engineering to improve plant resistance.