马铃薯in vitro 块茎发育的研究及其遗传转化受体系统的建立

研究了BAP在SLM→TIM、MA→TIM和切段诱导系统中与in vitro块茎发育的关系。在SLM→TIM系统中BAP没有促进块茎发育的作用;在MA→TIM系统中BAP具有促进块茎发育的作用;切段培养在20 ℃、8小时光照条件下则其块茎发育对BAP有依赖。系统地研究了光周期、蔗糖浓度和外源细胞分裂素在促进切段块茎发育方面的作用及其交互影响。确定了由切段诱导块茎的最佳培养条件以及用于BAP吸收代谢研究的实验条件。讨论了切段诱导系统在理论研究上的价值和生产上应用的前景。研究了无菌苗、长切段及切段BAP吸收运转代谢特点及其与块茎发育关系。马铃薯植物系统对BAP的吸收运转是需能代谢过程。BAP在植物系统运转性差与其在组织的代谢特点有关。在块茎诱导早期有标物质在小块茎或匍匐茎末端积累，这可能促使细胞分裂从而诱发块茎发育;但在成熟块茎中放射性物质浓度很低。外源细胞分裂素在切段或匍匐茎局部积累不是块茎发育的充分条件。在有利于块茎发育的条件下代谢早期BAP活性代谢产物含量明显地高于对照，代谢一定阶段后二种处理的切段BAP代谢谱趋于接近，这一代谢特点与BAP促进块茎发育的生理效应有关。运用同位素示踪技术研究了切段蔗糖吸收特点，外源细胞分裂素促进蔗糖在切段系统积累从而诱导体细胞储藏组织生化分化。短日照（黑暗）、较高浓度蔗糖、合适浓度BAP都有促进离体切段块茎发育作用，这几个因子在提高切段诱导水平方面具有协同效应。诱导早期切段中有块茎专一性糖蛋白Patatin痕量的存在。当切段发育了较大块茎后（诱导约15天）切段中Patatin含量明显增加。诱导3天切段系统中即有Patatin mRNA高水平地转录。诱导7天阶段Patatin mRNA含量迅速下降。块茎发育很可能是通过几种激素（包括块茎诱导因子）协同地对植物系统同化物尤其是蔗糖源库关系的调节而实现的。 建立了向马铃薯植物引入外源基因的受体系统。叶园盘与农杆菌（pGV2260:: pGV941）共培养。转化植株在选择培养基上培养大约3周即可从愈伤组织或直接从叶片边缘产生。在含有高浓度卡那霉素的培养基上转化植株大多表型正常，切段能够发育块茎，叶片能够形成愈伤组织。转化再生植株均含有NPT－II活性而未转化Desiree无菌苗没有NPT－II活性。Southern分析表明NPT－II基因已整合入转化植物基因组中。这一实验系统的建立为向马铃薯植物引进具有重要经济价值的外源基因创造了条件。

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from greater than or equal to 1000 mum diameter follicles

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its ac

IN-VITRO IMMUNOTOXICITY AND CYTOTOXICITY OF TRICHOSANTHIN AGAINST HUMAN NORMAL IMMUNOCYTES AND LEUKEMIA-LYMPHOMA CELLS

Trichosanthin (TCS) is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the effects of TCS on the viability of human peripheral blood immunocytess, on the proliferation of lymphocytes, and its cytotoxicity to twelve cell lines of lymphoma or leukemia had been observed. TCS at high concentration (>12.5 mu g/ml) affected the viability of human B lymphocytes, but not that of human peripheral blood mononuclear cells (PBMCs), T lymphocytes and granulocytes. Human peripheral blood-derived monocytes/macrophages were highly sensitive to TCS (ID50 at 1.70 mu g/ml). TCS suppressed lymphocyte proliferation stimulated by Concanavalin A (Con A) or lipopolysaccharide (LPS). Human T cell lines and macrophage cell lines were more sensitive (ID50 < 0.9 mu g/ml) to TCS than B cell lines and myeloid lines. These results suggest that selective cytotoxicity of TCS to human macrophages/monocytes may be implicated in anti-HIV activity, and that selectively killing some leukemia-lymphoma cells by TCS merit further evaluation in treatment of some lymphoma and leukemia.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.