条斑紫菜果胞形成与萌发的观察

条斑紫菜的果胞可由端部向藻体中央大量形成,并在母体萌发。

卫星搭载牛膝种子对其SP1植株胞果生物学特性的影响

利用返回式卫星搭载的中药牛膝种子为材料,与对照组牛膝种子相比较,对搭载后的牛膝种子SP1植株的种子生长发育过程进行了观测和分析,并对SP1植株获得的种子发芽及生长情况进行了进一步的观测。结果表明,经搭载后的航天组牛膝种子,SP1植株中出现不同程度的变异,特别是SP1植株的种子在发育过程中,粒色、百粒重、胞果大小与形状以及成熟时间等方面差异明显。此外,航天组与对照组SP1植株的种子在发芽势、发芽率及活力指数等指标上也表现出一定差异。说明卫星搭载可改变某些基因产物的表达量,从而表现出形态性状的显著差异。

杧果细菌性角斑病菌phd基因克隆与序列分析

由柑桔黄单胞菌杧果致病变种Xanthomonas citri pv.mangiferaeindicae引起的杧果细菌性角斑病是杧果上一种重要的细菌性病害,严重危害杧果产业发展。本试验获得了柑桔黄单胞菌杧果致病变种菌株Xcm003 phd基因的全序列。序列分析显示,phd基因编码85个氨基酸,其理论分子量9.68 kD,理论等电点为9.39,是一种亲水性蛋白,无信号肽,有6个磷酸化位点以及1个结构域。系统进化树结果显示,该基因的氨基酸序列与X.citri pv.mangiferaeindicae LMG941的Phd蛋白聚为一类。

利用SRAP分子标记及果肉汁胞色素分析鉴定柚子新品种黄金蜜柚

本研究利用SRAP分子标记进行分析鉴定,同时结合果肉汁胞色素分析、生物学性状及物候期观察,以确定黄金蜜柚的品种类型。通过208对SRAP引物对黄金蜜柚和琯溪蜜柚进行SRAP扩增,结果显示,两品种间的谱带基本相同,但均存在黄金蜜柚和琯溪蜜柚特有的谱带,这表明了两品种间在DNA水平上存在真实的遗传差异。对10份柚类品种的SRAP遗传相似性分析表明,黄金蜜柚与琯溪蜜柚的遗传距离最为接近,达到0.98,这从分子水平上证明了其亲缘关系非常紧密,同系列的琯溪蜜柚、黄金蜜柚、红肉蜜柚和三红蜜柚4个柚类品种间的遗传相似系数均较高。另外黄金蜜柚在汁胞色素种类及含量、花柱头颜色和成熟期等方面均明显区别于琯溪蜜柚。研究结果充分表明了黄金蜜柚是一个不同于其它柚类的优特柚类新品种。

Study on Improving the Internal Quality of Tobacco Stems by Using Pectinase

[Objective] This study aimed to reduce the content of cell wall materials in tobacco stems and improve their internal quality. [Method] Pectinase was used to decompose cell wall materials in tobacco stems, followed by determination of the contents of four cell wall materials, six routine chemical components, as well as aroma constituents. [Result] Pectinase could effectively reduce the contents of cell wall materials in tobacco stems, with the largest decrease of 6.84%; after pectinase treatment,the content of reducing sugar in tobacco stems increased obviously, and the contents of total sugar, potassium ion, chloride ion and total nitrogen increased to varying degrees, of which the contents of potassium ion and reducing sugar displayed upward trends with the increase of pectinase concentration. Pectinase treatment significantly increased the contents of Maillard reaction products, with the most increase of 67.2%;the contents of carotenoid degradation products, phenylalanine degradation products and neophytadiene all increased to varying extents, and the contents of both Maillard reaction products and phenylalanine degradation products revealed ascending trends with the increase of pectinase concentration. [Conclusion] Pectinase treatment can effectively decompose cell wall materials in tobacco stems, improve routine chemical constituents, and increase the contents of aroma constituents.

In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits

The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-3-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-3-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd = 2.9 nmol/L) and the other with lower affinity ( Kd = 71.4 nmol/L). Phaseic acid, 2-trans-4-trans-ABA or cis-trans-(-)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.

Activities, Quantitative Changes and Subcellular Localization of α-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

Research on Anti-bacterial Effects of 5 Kinds of Chinese Herb Extracts on Ameromonas hydrophila in vitro

[Objective] The aim was to study the anti-bacterial effects of 5 kinds of Chinese herb extracts on Ameromonas hydrophila in vitro. [Method] in vitro anti-bacterial effects of 5 kinds of Chinese herb extracts like Galla Chinensis,Syzygium aromaticum,Salvia miltiorrhiza,Punica granatum L. and Terminalia chebula Retz on Ameromonas hydrophila were studied; furthermore,cure rates of the Chinese herb extracts with better anti-bacterial effects were determined to find out the optimal drug dosage. [Result] Under the same experimental conditions,Galla Chinensis,Punica granatum and Terminalia chebula Retz had relatively strong anti-bacterial effects on Ameromonas hydrophila,among them the anti-bacterial effect of Galla Chinensis was significantly higher than those of the others （P0.05）. The optimal treatment dose of Galla Chinensis treating bacterial septicemia caused by Aeromonas hydrophila was that they were treated with medicated bath for 40 min by 0.5 mg/ml Galla Chinensis extract,and the cure rate was 100%. [Conclusion] The research provides a scientific drug basis for the control and prevention of outbreak bacterial diseases of fish.

Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.

Deletion of 93 bp Far-upstream Fragment of Rice Cytosolic Fructose- 1, 6-Bisphosphatase Promoter Completely Alter Its Expression Pattern

The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements responsible for the regulation of mesophyll_specific expression, the 5′ flanking regions of -1 195 bp, -1 102 bp, -768 bp, and -644 bp upstream of the translation initiation ATG codon were fused to the reporter gene encoding β_glucuronidase (GUS) and transferred to rice via particle bombardment. Analysis of the 5′ promoter deletions identified that a 93 bp fragment between -1 195 bp and -1 102 bp is essential for directing mesophyll specific expression. High constitutive expression of GUS reporter gene was found in the -768 deletion lines and another two deletion series. These results indicate the great potential utility of the promoter in rice biotechnology.

Genetic regulation of programmed cell death in Drosophila

Programmed cell death plays an important role in maintaining homeostasis during animal development, and has been conserved in animals as different as nematodes and humans. Recent studies of Drosophila have provided valuable information toward our understanding of genetic regulation of death. Different signals trigger the novel death regulators rpr, hid, and grim, that utilize the evolutionarily conserved iap and ark genes to modulate caspase function. Subsequent removal of dying cells also appears to be accomplished by conserved mechanisms. The similarity between Drosophila and human in cell death signaling pathways illustrate the promise of fruit mes as a model system to elucidate the mechanisms underlying regulation of programmed cell death.

Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15

AIM: Ursolic acid (UA) and oleanolic acid (OA) are triperpene acids having a similar chemical structure and are distributed wildly in plants all over the world. In recent years, it was found that they had marked anti-tumor effects. There is little literature currently available regarding their effects on colon carcinoma cells. The present study was designed to investigate their inhibitory effects on human colon carcinoma cell line HCT15. METHODS: HCT15 cells were cultured with different drugs. The treated cells were stained with hematoxylin-eosin and their morphologic changes observed under a light microscope. The cytotoxicity of these drugs was evaluated by tetrazolium dye assay. Cell cycle analysis was performed by flow cytometry (FCM). Data were expressed as means +/-SEM and Analysis of variance and Student' t-test for individual comparisons. RESULTS: Twenty-four to 72 h after UA or OA 60 micromol/L treatment, the numbers of dead cells and cell fragments were increased and most cells were dead at the 72nd hour. The cytotoxicity of UA was stronger than that of OA. Seventy-eight hours after 30 micromol/L of UA or OA treatment, a number of cells were degenerated, but cell fragments were rarely seen. The IC(50) values for UA and OA were 30 and 60 micromol/L, respectively. Proliferation assay showed that proliferation of UA and OA-treated cells was slightly increased at 24h and significantly decreased at 48 h and 60 h, whereas untreated control cells maintained an exponential growth curve. Cell cycle analysis by FCM showed HCT15 cells treated with UA 30 and OA 60 for 36 h and 72 h gradually accumulated in G(0)/G(1) phase (both drugs P【0.05 for 72 h), with a concomitant decrease of cell populations in S phase (both drugs P【0.01 for 72 h) and no detectable apoptotic fraction. CONCLUSION: UA and OA have significant anti-tumor activity. The effect of UA is stronger than that of OA. The possible mechanism of action is that both drugs have an inhibitory effect on tumor cell proliferation through cell-cycle arrest.

The Drosophila ovary: an active stem cell community

Only a small number of cells in adult tissues （the stem cells） possess the ability to self-renew at every cell division, while producing differentiating daughter cells to maintain tissue homeostasis for an organism＇s lifetime. The Drosophila ovary harbors three different types of stem cell populations （germline stem cell （GSC）, somatic stem cell （SSC） and escort stem cell （ESC）） located in a simple anatomical structure known as germarium, rendering it one of the best model systems for studying stem cell biology due to reliable stem cell identification and available sophisticated genetic tools for manipulating gene functions. Particularly, the niche for the GSC is among the first and best studied ones, and studies on the GSC and its niche have made many unique contributions to a better understanding of relationships between stem cells and their niche. So far, both the GSC and the SSC have been shown to be regulated by extrinsic factors originating from their niche and intrinsic factors functioning within. Multiple signaling pathways are required for controlling GSC and SSC self-renewal and differentiation, which provide unique opportunities to investigate how multiple signals from the niche are interpreted in the stem cell. Since the Drosophila ovary contains three types of stem cells, it also provides outstanding opportunities to study how multiple stem cells in a given tissue work collaboratively to contribute to tissue function and maintenance. This review highlights recent major advances in studying Drosophila ovarian stem cells and also discusses future directions and challenges.

Therapeutic effectiveness of echo-guided percutaneous radiofrequency ablation therapy with a LeVeen needle electrode in hepatocellular carcinoma

AIM： To investigate the results of radiofrequency ablation （RFA） in obtaining the necrosis of hepatocellular carcinoma （HCC） in cirrhotic patients and to assess the results of RFA in relation to recurrence of HCC and survival of the treated patients.METHODS： Fifty-six consecutive cirrhotic patients with 63 HCCs were treated with RFA between May 2000 and May 2004. The diameter of the HCCs ranged from 1 cm to 5 cm （mean 2.8 cm）. In all cases RFA was performed with percutaneous approach under ultrasound guidance using expandable needle electrode （LeVeen needle）. Treatment efficacy and recurrence were evaluated with dual-phase spiral computed tomography （CT）.RESULTS： Complete necrosis after single or multiple treatment was achieved in 96.8% （61/63） tumors. We observed recurrence after complete necrosis in 23 patients （41%） during a mean follow-up of 32.3 months. The recurrences were local in 2 patients （8.6%） and in different segments in 21 （91.4%）. Major complications occurred in 3 patients （4%）. During follow-up period, 32 （57.1%） patients died; 15 due to progression of HCC, 11 from liver failure, 3 from esophageal varices bleeding and 3 from the causes not related to liver disease.CONCLUSION： RFA with LeVeen needle is an effective and safe treatment for HCC 〈 5cm in cirrhotic patients. It has yet to be established how far this treatment influences the survival rate of patients. It becomes important to establish treatments to prevent recurrences in different segments, such as interferon therapy.

Programmed cell death features in apple suspension cells under low oxygen culture

Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmos-phere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Pro-toplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidyl-serine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

Inhibition of pancreatic carcinoma cell growth in vitro by DPC4 gene transfection

AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas,and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro.METHODS: The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry.Furthermore,we constructed retroviral vectors containing DPC4,which then infected the pancreatic carcinoma cell line BxPC-3.Cell growth in vitro after being infected was observed,and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay.RESULTS: The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens,compared to 40% (16/40) in adenocarcinoma specimens.The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four,whereas it was all positive expression in normal tissues.There was a significant difference of DPC4 expression between them.The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection,and could inhibit cell growth in vitro.Rather,DPC4 could decrease VEGF mRNA transcription levels.CONCLUSION: The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma.The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells,and down-regulate the level of VEGF.

Tudor and its domains: germ cell formation from a Tudor perspective

In many metazoan species, germ cell formation requires the germ plasm, a specialized cytoplasm which often con-tains electron dense structures. Genes required for germ cell formation in Drosophila have been isolated predominantlyin screens for maternal-effect mutations. One such gene is tudor (tud); without proper tud function germ cell formationdoes not occur. Unlike other genes involved in Drosophila germ cell specification tud is dispensable for other somaticfunctions such as abdominal patterning. It is not known how TUD contributes at a molecular level to germ cell forma-tion but in tud mutants, polar granule formation is severely compromised, and mitochondrially encoded ribosomal RNAsdo not localize to the polar granule. TUD is composed of 11 repeats of the protein motif called the Tudor domain. Thereare similar proteins to TUD in the germ line of other metazoan species including mice. Probable vertebrate orthologuesof Drosophila genes involved in germ cell specification will be discussed.

The Biological Effect of Hepsin on the Proliferation and Invasion of PC-3 Prostate Cancer Cells

OBJECTIVE Recent studies have shown that hepsin, a type of transmembrane serine protease, is highly upregulated in prostate cancer, but, little is known about its role in progression and invasion of this cancer. We constructed a hepsin-expressing plasmid and transfected it into PC-3 cells to investigate the effect of the hepsin gene on the biological behavior of the PC-3 cells. METHODS Plasmid pHepsin-IRES2 was transfected into prostate cancer PC-3 cells using Fugene6, and the cells with stable hepsin expression were screened and selected with Zeocin (600 mg/L). The hepsin mRNA level was measured by real-time PCR and the growth curve of the PC-3-transfected cells assessed using MTT and BrdU assays. A Boyden chamber was used to examine the difference in invasion and metastases between transfected and non-transfected cells. RESULTS The hepsin mRNA level in pHepsin-IRES2 transfected -PC-3 cells was significantly higher than that found in the control PC -3 cells. While the growth curve of the hepsin gene transfected PC -3 cells showed that there was no significant effect on proliferation, the invasive ability of the pHepsin-IRES2 transfected PC-3 cells, as compared with control cells, was significantly increased (P<0.05). CONCLUSION The results suggest that even though hepsin has no effect on the proliferation of prostate cancer PC-3 cells, it does promote cellular invasion and metastasis.Therefore hepsin may have a role in the development of prostate cancer.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.