雨生红球藻的新繁殖方式及其藻蓝蛋白的合成

雨生红球藻(Haematococcus pluvialis)是一种能够合成强抗氧化剂-虾青素的单细胞绿藻,然而由于其生长速率缓慢,制约了虾青素产量的提高。在对雨生红球藻细胞周期的研究中,首次发现了雨生红球藻在高温和低光照条件下的一种特殊繁殖方式,该繁殖方式比已知的营养繁殖和无性繁殖速度快。繁殖过程中厚壁孢子萌发产生大量的小细胞,并且这些小细胞中含有以前在绿藻中从未发现的捕光色素蛋白—藻蓝蛋白(最大吸收波长λmax=621 nm,最大荧光发射波长λmFax=643 nm)。这一发现对于提高天然虾青素的产量和藻蓝蛋白的生产以及藻类的进化有着重要的意义。     

转光膜在雨生红球藻养殖上的应用

通过对雨生红球藻在不同光质条件下生长的比较,确定了红色光有利于藻生长,进而用2.5 L气升式光照反应器在转光膜及普通PE膜下培养藻进行对比,结果显示雨生红球藻生物量、色素、光合活性等几项生物指标在转光膜条件下明显高于普通PE膜. 在气升式反应器内培养的藻细胞,接种9 d,虾青素含量可达3.57 mg/L,叶绿素浓度达到12.42 mg/L,干重提高8.8%以上.     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

雨生红球藻内生蓝藻转变为一种新的绿藻

雨生红球藻细胞释放的无类囊体内生蓝藻TDX16在强光条件下其细胞内细胞器依次形成。首先细胞中新合成的电子致密物质和淀粉状物质与原核细胞质分离,从而形成2个区室:即收缩的原核细胞质及其周边充满电子致密物质和淀粉状颗粒的环型区域。然后收缩的原核细胞质发育成含有光合片层,蛋白核和淀粉颗粒的超大叶绿体;而环型区域的物质则组装成线粒体和细胞核。最后叶绿体产生的球状体释放出内含物后变成液泡,而内含物则积累形成真核细胞质。伴随着叶绿体的形成,TDX16开始在光合片层上合成新光合色素如叶绿素b,而少量的藻蓝蛋白则残留在其细胞膜上,由此TDX16转变成含有叶绿素b和藻蓝蛋白的特殊绿藻。TDX16的这一转变在细胞生物学的研究方面具有重要的意义,并且其形成的绿藻在生物化工应用方面具有潜在的应用价值。     

雨生红球藻合成虾青素过程中分泌铵离子的研究

雨生红球藻在在缺氮(NF)的条件下,随着虾青素的合成,肽链内切酶(EP)的活性上升而蛋白质含量下降,天冬酰氨酸含量在前4天增加然后又减少,铵离子浓度则持续上升。相比之下,在氮充足(NR)的培养液中,藻细胞不合成虾青素,蛋白质和天冬酰氨酸的含量以及EP的活性基本稳定,培养液中也没有检测到铵离子。上述结果表明:①降解的蛋白质为虾青素的合成提供了碳源,②EP参与了蛋白质的降解反应,③为避免铵离子的毒害作用,蛋白质降解所产生的部分氨临时贮存在天冬酰氨酸中,而其余的则分泌到细胞外。     

强光照对雨生红球藻合成虾青素的影响

在不同的光照强度下研究了雨生红球藻细胞内虾青素的合成与初级代谢的关系.在强光(HL)和中等强度(ML)的光照条件下,雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶 (Rubisco)和硝酸还原酶(NR)的活性第1天大幅度提高,2天后又迅速下降.与此同时,硝酸盐浓度也快速降低.当虾青素在第4天(HL)和第6天(ML)开始合成时, HL中Rubisco和NR活性以及NO3-浓度分别下降了75.5%,71.5% 和96.2%,而ML中则下降了76.5%,74.7% 和94.3%.相比之下,在低光照(LL)条件下,实验结束时三个指标仅下降了25.9%,29.8% 和56.8%,细胞中没有虾青素积累.结果表明强光提高了Rubisco 和 NR活性,导致硝酸盐浓度迅速降低而最终又抑制了这两种酶的活性,造成雨生红球藻光合作用效率下降即"碳饥饿".在此状态下,为了生存,细胞内合成虾青素的相关基因被激活,藻细胞开始合成并积累虾青素.     

碳和氮代谢被抑制诱导雨生红球藻细胞内虾青素的合成

为研究雨生红球藻(Haematococcus pluvialis)合成虾青素的机理,文中分析了不同诱导条件下藻细胞内氮和碳代谢的变化。结果表明:强光照(HL)、添加乙酸钠(AA)、缺氮(NF)和缺磷(PF)都直接或间接地影响了雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶(Rub isco)和硝酸还原酶(NR)的活性,导致2种酶的活性大幅度下降。只有当Rub isco和NR的活性降到非常低的水平时,藻细胞才开始合成虾青素。与此相反,对照(CK)中这2种酶的活性一直较高,但细胞内没有虾青素积累。由于Rub isco和NR是雨生红球藻碳代谢和氮代谢的关键酶,因此碳和氮代谢被抑制是诱导雨生红球藻合成虾青素的原因。     

盐胁迫诱导雨生红球藻合成虾青素的机理

为研究盐胁迫诱导雨生红球藻合成虾青素的机理,分析了在添加氯化钠(HS)和未添加氯化钠(CK)的培养液中,细胞内氮和碳代谢的变化。结果表明:HS中硝酸还原酶(NR)和1,5-二磷酸核酮糖羧化酶(Rub isco)活性迅速下降。虾青素在第4天开始合成时,二者分别降至初始值或最高值的46.5%和25.7%。相比之下,在对照(CK)中,NR和Rub isco活性仍然很高,仅下降了26.1%和25.6%,细胞内没有虾青素积累。上述数据表明盐胁迫条件下NR活性被抑制,细胞内氮源供应不足(氮饥饿)并进一步抑制了Rub isco的合成,导致CO2固定量减少(碳饥饿)。为了生存,藻细胞开始合成虾青素。     

Concomitant NH_4~+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin syn-thesis in the cultures of high light and nitrogen-free (HF), high light and nitrogen-repletion (HR), and low light and nitrogen-free (LF), (1) endopeptidase (EP) activities increased along with decrease in protein content, (2) aspar-agine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, (3) ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, (LR), the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas (O2 and N2), astaxan-thin content increased as the protein level decreased. These results strongly suggest that (1) the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, (2) endopeptidase was involved in the degradative process, (3) for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.     

换热器中介质温度对超声波传播特性的影响

在超声波防除垢试验台上,通过实验研究温度对除垢超声波传播特性的影响。保持液体流速、超声波发射频率和功率恒定,测量不同温度下换热管测点1和测点2处声强的大小,分析超声波声强随温度变化的规律,确定介质温度对换热管道内超声波传播特性的影响。结果表明:实验温度在25～55℃范围内时,超声波衰减随温度的升高而减小;实验温度在55～75℃范围内时,超声波衰减随温度的升高而增大。     

The Effects of Synthetic Oligopeptide Derived from Enamel Matrix Derivative on Cell Proliferation and Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Enamel matrix derivative (EMD) is widely used in periodontal tissue regeneration therapy. However, because the bioactivity of EMD varies from batch to batch, and the use of a synthetic peptide could avoid use from an animal source, a completely synthetic peptide (SP) containing the active component of EMD would be useful. In this study an oligopeptide synthesized derived from EMD was evaluated for whether it contributes to periodontal tissue regeneration. We investigated the effects of the SP on cell proliferation and osteoblast differentiation of human mesenchymal stem cells (MSCs), which are involved in tissue regeneration. MSCs were treated with SP (0 to 1000 ng/mL), to determine the optimal concentration. We examined the effects of SP on cell proliferation and osteoblastic differentiation indicators such as alkaline phosphatase activity, the production of procollagen type 1 C-peptide and osteocalcin, and on mineralization. Additionally, we investigated the role of extracellular signal-related kinases (ERK) in cell proliferation and osteoblastic differentiation induced by SP. Our results suggest that SP promotes these processes in human MSCs, and that ERK inhibitors suppress these effects. In conclusion, SP promotes cell proliferation and osteoblastic differentiation of human MSCs, probably through the ERK pathway.     

Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.