Ca2+ triggers a novel clathrin-independent but actin-dependent fast endocytosis in pancreatic beta cells

The existence of clathrin-independent recycling of secretory vesicles has been controversial. By combining patch-clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic β cells, both of which require GTP and dynamin. The fast one is a novel clathrin-independent but actin-dependent endocytosis that is triggered by high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). Large fluorescent dextran (10 nm in diameter) was able to be internalized by this pathway, indicating that it was not likely to be 'kiss and run'. The slow endocytosis is a clathrin-dependent process in which actin plays a complementary role. For the first time, we show that the rate constants for both types of endocytosis exhibit supralinear dependence on increase in [Ca²⁺]_i. Compared with the slow endocytosis, higher [Ca²⁺]_i level was required to fully accelerate the fast one, indicative of distinct Ca²⁺ sensors for different endocytosis. In the end, we show that physiologically relevant stimulation induces clathrin-independent endocytosis in intact β cells, implying that it may contribute to the normal recycling of secretory vesicles in vivo .     

Molecular characterization of myostatin in black seabream, Acanthopagrus schlegelii.

Myostatin is a negative regulator of skeletal muscle growth and has a potential application in aquaculture. The black seabream myostatin gene was cloned and sequenced. It had three exons encoding a protein of 382 amino acids. A 90 bp 5'-untranslated region (UTR) and a 536 bp 3'-UTR were obtained by RACE. Four microsatellite sequences, a (CAG)9, a (TC)12, a (CA)16 repeat and an "imperfect" (CA)25 microsatellite, were found in the myostatin. Two introns were 329 and 742 bp in length, respectively. The deduced amino acid sequence of the myostatin had a putative amino terminal signal sequence, a TGF-beta propeptide domain, a RXXR proteolytic processing site, a TGF-beta domain, and 12 conserved cysteine residues. The myostatin gene was expressed in four of the examined ten tissues and organs. The expression of myostatin was the strongest in the skeletal muscle and brain, intermediate in the eye, and low in the heart.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

中国森林生态系统服务功能价值评估

森林作为陆地生态系统的主体,在全球生态系统中发挥举足轻重的作用,其服务功能价值的评估是研究的一个热点。本研究根据全国第5次资源清查资料(1994~1998年)及Costanza等人的计算方法估算了我国森林生态系统八项服务功能的总价值为30601.20×108yuan/a,其中间接价值是直接经济价值的14.94倍;在我国森林生态系统中,单位面积的各种森林生态系统所提供的年平均价值为23095.25yuan/(hm2·a)。在森林涵养水源、固碳释氧、营养物质循环、净化空气和保护土壤五项服务功能中,热带雨林、季雨林单位面积所提供的价值量最大为32417.45yuan/(hm2·a),温带荒漠单位面积所提供的价值最小为12507.90yuan/(hm2·a);在森林生态系统各项服务功能价值的贡献之当中,其大小顺序依次为:固碳释氧>净化空气>土壤保持>涵养水源>养分循环>林木、林副产品〉维持生物多样性〉森林游憩。该研究的目的在于尽快将自然资源和环境因素纳入国民经济核算体系而最终为实现绿色GDP提供基础,为我国可持续发展的政策与生态环境保护提供科学依据。     

大黄鱼MSTN成熟肽区基因的原核表达

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。     

Bulk-like endocytosis plays an important role in the recycling of insulin granules in pancreatic beta cells

Although bulk endocytosis has been found in a number of neuronal and endocrine cells, the molecular mechanism and physiological function of bulk endocytosis remain elusive. In pancreatic beta cells, we have observed bulk-like endocytosis evoked both by flash photolysis and trains of depolarization. Bulk-like endocytosis is a clathrin-independent process that is facilitated by enhanced extracellular Ca²⁺ entry and suppressed by the inhibition of dynamin function. Moreover, defects in bulklike endocytosis are accompanied by hyperinsulinemia in primary beta cells dissociated from diabetic KKAy mice, which suggests that bulk-like endocytosis plays an important role in maintaining the exo-endocytosis balance and beta cell secretory capability.