Fen zi xi bao sheng wu xue bao = Journal of molecular cell biology最新文献

Antisense RNAs have often been used to study functions of human genes. In order to obtain the effective antisense fragments, researchers always have to face repeated works including cell culture and transfection etc. Here we chose yeast, Saccharomyces cerevisiae, as a model organism to explore the simple and effective way to obtain antisense fragments targeting human genes. This single-cell eucaryote divides rapidly, can be cultured easily just as E. coli, and possesses many biological processes similar to mammalian. Thus, it is the best choice for the study focus on human gene transcription and other biological processes. Human Elongator complex was found to be very similar to the yeast Elongator both in composition and its interaction with RNA polymerase II. The elp3 subunit of human Elongator can significantly complement the growth defects of Yeast elp3 Delta Strain. So we used yeast complementation systems to select the antisense fragment targeting help3. Three different fragments including full length (a1 644), HAT-deletion (a1 107) and HAT(a450) sequence of help3 were amplified and inserted into pRS313 or pRS314 to construct antisense plasmids pRS313a1 644, pRS314a1 107 and pRS314a450. Through the sensitive assay and RT-PCR detection of help3 and ssa3 expression in yeast, we approved that the HAT-deletion fragment (a1 107) repressed the expression of cotransformed help3 significantly and reduced the complementation ability of help3 dramatically in yeast. Then the a1 107 antisense mammalian expressing plasmid was constructed and transfected into Hela cells. Northern blot assay indicated that a1 107 significantly reduced the expression of help3 in HeLa cells. So we regard that cotransforming targeting human gene and its antisense fragments into yeast and obtaining the significant reduction expression of targeting human gene is a simple strategy for the selection of antisense fragment targeting human genes.

The recombinant adenoviruses which can overexpress fosb and Delta2Deltafosb proteins were produced. Primary calvarial osteoblasts from new born CD1 mice were infected with those adenoviruses by different proportion of Ad-Delta2Deltafosb and induced by 5 mmol/L beta-glycerophosphate, 50 microg/ml ascorbic acid and 10 nmol/L dexamethasone for 23 days and 29 days, respectively. The results by Alizarin Red Staining showed that the primary osteoblasts overexpressing Delta2Deltafosb protein formed more nodus than the control cells and the nodus in overexpressing Delta2Deltafosb protein osteoblasts were smaller than those in control cells on day 23. Differentiation of the primary osteoblasts overexpressing Delta2Deltafosb protein increased more than that of the control cells on day 29. It is an evidence for the reason that bone density of Delta2Deltafosb transgenic mice was markedly increased throughout the skeleton in new born and mature mice.

By using genetic transformation of Agrobacterium rhizogenes and liquid culture, induction and culture conditions for Solanum nigrum L. var. pauciflorum Liou hairy roots and its solasodine production and consumption changes of N resource and calcium in the medium during liquid culture were investigated. The results showed that hairy roots could be initiated from the cut edges of leaf explants 5 days after inoculation with the strain of A. rhizogenes ATCC15834. The percentage of rooted leaf explants 25 days after infection was more than 90%. Hairy roots could grow rapidly on solid or liquid growth regulator-free MS medium. The PCR amplification of rol genes and virC gene showed that rol genes of Ri plasmid of A. rhizogenes were integrated in the genome of transformed hairy roots of S. nigrum L var pauciflorum. The hairy roots could produce medicinal secondary metabolites solasodine and the amount of solasodine in the hairy root cultures reached a level of 582.05 microg/g dry weight and was 1.31 times as much as those in the untransformed roots. The hairy roots grew very slowly in 0-5 days in the liquid medium, then, very fast from 5 to 15 days. During liquid culture, NO3(-) and NH4(+) in the medium were gradually absorbed and utilized by hairy roots. NH4(+) -N of the medium was used up at day 15 of the culture, while NO3(-) in the medium was not used up, the content of which was 44.7% of the initial amount. Ca2+ of the medium was gradually absorbed and utilized during liquid culture and it was not used up at day 25, the content of which was still 43.5% of the initial amount. The results presented here had provided the possibilities on how to prepare optimum medium for large scale cultivation and production of solasodine from S. nigrum L. var. pauciflorum hairy roots.

To observe the expression of mitogen activated protein kinase phosphatase-1 (MKP-1) and phosphorylation of extracellular signal-regulated kinases (P-ERK) of spinal cord in acute contusive spinal cord injury model, 20 Sprague Dawley rats were divided into two groups at random, Ten rats' spinal cord contusive injuries were produced by using modified Allen's method (using a weight-drop device) after the T10 spinous process and the corresponding vertebral lamina were removed as experimental group. The rest 10 rats received only T10 laminectomies and didn't injury the spinal cord as Sham-operated control group. The injury spinal cord was carried out respectively in two groups at 12h after injury. Pathological alterations were detected by H-E stain. The expression of MKP-1 and P-ERK were analyzed by immunohistochemistry and Western blot analysis. In Sham-operated control group, the micro structure of spinal cord was normal. The pathological alterations were very apparent in the damaged spinal cord area in the experiment group. We also found MKP-1 expression of spinal cord was decreased while P-ERK was increased in experimental group when compared with Sham-operated control group (P<0.01). The results suggested that acute contusive spinal cord injury down-regulated the expression of MKP-1 and up-regulated the PERK, which might play a role in the pathophysiological of spinal cord injury.

AP2/ERF is a large family of transcription factors in plant. Genes in the AP2/ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. Two AP2/ERF family transcriptional regulators (BnaERFB3-1 and BnaERFB3-2) were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana AP2/ERF-B3 subfamily as probe. Based on the sequences of BnaERFB3-1 and BnaERFB3-2, we isolated the BnaERFB3-1-Hy15 gene and BnaERFB3-2-Hy15 gene from winter and spring type B. napus L. cv Huyou15 by RT-PCR and PCR using cDNA and DNA as template. DNA sequencing and analyzing indicated that there was only one amino acid residue difference between BnaERFB3-1 and BnaERFB3-1-Hy15, BnaERFB3-2 and BnaERFB3-2-Hy15, respectively. No intron localized on the two genes from Huyou15. Then, deduced amino acid sequence, composition, hydrophobicty and hydrophilicity, physical and chemical characterization, phylogenetic tree, conserved domain sequences, function domain, molecular modeling, and folding state were predicted and analyzed. BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were hydrophilic protein. The two proteins and AtERF5 have similar three-dimension structure. The disordered residues of protein BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were higher than that of AtERF5. BnaERFB3-1 was mainly expressed in seed, while BnaERFB3-2 was mainly expressed in root. Moreover, those genes were successfully constructed into the recombinant plasmids of plant expression vector and yeast expression vector, which established a base for transformation of oilseed and studies of those genes function in abiotic stresses of B. napus.

Wheat-Haynaldia villosa chromosome substitution line (6A/6V) and translocation lines (6DL/6VS, 6AL/6VS) were obtained through hybridization of H. villosa with powdery mildew susceptible cultivated wheat. Substitution line and translocation lines contain V chromosome or the chromosome short arm (VS) of H. villosa. They are resistant to powdery mildew. In this study, mitochondrial proteome changes were analyzed by using substitution line (6A/6V), translocation line (6DL/6VS) as experimental materials in order to studying the effects of V chromosome on the mitochondrial proteome and related to powdery mildew resistance. The results indicated that 16 new mitochondrial protein spots (spot1, 22kDa/PI8.5; spot2, 31 kDa/PI 7.5; spot3, 28 kDa/PI 7.0; spot4, 31 kDa/PI 6.5; spot5, 40 kDa/PI 7.5; spot6, 40 kDa/PI 7.4; spot7, 80 kDa/PI 8.4; spot8, 50 kDa/PI 7.5; spot9, 60 kDa/PI 7.3; spot10, 65 kDa/PI 6.6; spot11, 65 kDa/PI 6.6; spot12, 73 kDa/PI 7.5; spot13, 73 kDa/PI 7.7; spot14, 46 kDa/PI 7.4; spot15, 46 kDa/PI 7.3; spot16, 38 kDa/PI 6.3) were produced and 7 mitochondrial protein spots (spot1, 40 kDa/PI 7.5; spot2, 43 kDa/PI 7.6; spot3, 48 kDa/PI 7.5; spot4, 42 kDa/PI 8.0; spot5, 43 kDa/PI 7.5; spot6, 32 kDa/PI 4.8; spot7, 40 kDa/PI 5.5) were absent in substitution line, 7 new mitochondrial protein spots (spotl, 43 kDa/PI 6.3; spot2, 60 kDa/PI 6.5; spot3, 60 kDa/PI 6.4; spot4, 65 kDa/PI 7.5; spot5, 55 kDa/PI 8.2; spot6, 31 kDa/PI 8.0; spot7, 43 kDa/PI 8.0) were produced and 6 mitochondrial protein spots (spot1', 66 kDa/PI 8.3; spot2', 58 kDa/PI 8.5; spot3', 36 kDa/PI 7.0; spot4', 48 kDa/PI 7.7; spot5', 48 kDa/PI 6.8; spot6', 43 kDa/PI 6.2) were absent in translocation line. These experimental results suggest that V chromosome or VS of H. villosa can obviously lead mitochondrial proteome changed. These changes may be associated with resistant to powdery mildew of substitution line and translocation line.

The development of secretory canals in vegetative organs of Bupleurum chinense DC. and the accumulation of essential oils were investigated by transmission electron microscopy (TEM) analysis. The secretion mechanism of the essential oil was also discussed. The results indicate that the plastid, ground substances of cytoplasm and mitochondria took part in the biosynthesis of oil or oil precursor. The endoplasmic reticulum involved in the transport of essential oil to the secretory lumen. At latter stages of development of secretory cells, numerous different sized vesicles fused with the plasmalemma along the boundary between two neighbor secretory cells and secreted the substance into the wall. Thus, the wall between two neighbor secretory cells near the lumen became loosely structured. Then, the wall lined the lumen near two neighbor secretory cells extruded numerous grey vesicles with various sizes on the side facing the lumen, and released these vesicles into the lumen. As a result, the manner of secretion in secretory canals of Bupleurum chinense DC. appeared to be exocytosis.

To study the expression and function of protein metabolism, folding, transport, localization and assembly-associated genes in rat liver regeneration (LR) at transcriptional level, we obtained above genes from databases and scientific articles, detected their expression profiles in rat LR using Rat Genome 230 2.0 array, and determined liver regeneration-associated genes by comparing the partial hepatectomy (PH) group with sham operation (SO) group. Totally 1147 genes were preliminarily confirmed to be LR-associated. The results from the chip detection demonstrated that genes involved in the above biological processes were mostly up-regulated in rat LR; protein metabolism-participating genes were initially expressed mainly at 0.5-1 h and 16-30 h following PH; protein degradation-accelerating genes outnumbered protein accumulation-promoting genes between 0.5-12 h, whereas the latter were more than the former during 16-48 hours; protein synthesis-involved genes were more frequently up-regulated at 16,24,42 and 66 h, especially at 42 h; up-regulation of protein degradation-associated genes dominated almost during the whole period of LR, especially at forepart and prophase; the up-regulated protein folding-associated genes were predominant than down-regulated at 2, 16-24, 42, 66, 72 and 168 h, especially at 66 h; protein transport and localization-associated genes were predominantly up-regulated during the whole period of LR, especially at 66 h; and most of protein complex assembly-associated genes were up-regulated before 96 h, especially at 12 h. it was inferred according to the above analysis that protein synthesis was enhanced at metaphase of LR, and the activities of protein degradation, folding, transport, localization and assembly were vigorous almost during the whole period of LR.

To research if the vascular smooth muscle cells (VSMCs) from human umbilical artery undergo osteoblast differentiation spontaneously in vitro. The growth curve of vascular smooth muscle cells from human umbilical artery was obtained by MTT method. The course of multicell nodule formation spontaneously by VSMCs was observed morphologically. The apoptosis of VSMCs in the nodules was detected by Hoechst 33258 and TUNEL methods respectively. The expression of alkaline phosphotase in the nodules was detected by immunohistochemical method. And the calcification was studied with transmission electron microscope and by alizarin red S respectively. We found that the umbilical artery smooth muscle cells confluenced after 7 days of passage and exhibited typical "hill and valley" pattern under light microscope. The cells grew into aggregation and formed nodules at the "hill" region with culture-time prolongation. After 4-5 weeks culture, these nodules built up and calcified spontaneously. We also found alkaline phosphotase expression and apoptosis of VSMCs in these nodules at the same time. We conclude that the vascular smooth muscle cells from human umbilical artery just like from aortic artery can undergo osteoblast differentiation spontaneously in vitro, and apoptosis participate this procedure probably.

Serine/arginine protein kinases are specific kinase family for phosphorylating SR protein regulating alternative splicing of SR protein and its distribution, localization in the nucleus. However, it is unclear how Physarum Polycephalum Serine/Arginine Protein Kinase(PSRPK) functions in the cells. In order to study its function, Oligonucleotides for transcribing siRNAs were designed and inserted into pSIREN-RetroQ vector to construct pSIREN-PSRPK-1, pSIREN-PSRPK-2, pSIREN-PSRPK-3, pSIREN-PSRPK-4, pSIREN-PSRPK-5 for expressing siRNAs targeting at PSRPK, as well as the negative control pSIREN-PSRPK-Neg. The PSRPK cDNA amplified by PCR was inserted into the pDsRed-N1 vector to construct a pPSRPK-DsRed plasmid. After the pPSRPK-DsRed was co-transfected into HEK293 cell with recombinant siRNA expression plasmids respectively, the PSRPK-DsRed fusion fluorescent protein was observed under fluorescent microscope after 72 hours co-transfection. The results indicated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 were able to inhibit the expression of PSRPK-DsRed fusion fluorescent protein efficiently. RT-PCR and Northern dot blot analysis further demonstrated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 can effectively inhibit PSRPK expression, which accorded with the results under the fluorescent microscope.