Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (∼ ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.     

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.     

Topoisomerase II binds importin alpha isoforms and exportin/CRM1 but does not shuttle between the nucleus and cytoplasm in proliferating cells

Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.     

Study of the mechanism of small GTPase Ras-dva intracellular localization

An analysis of amino acid sequences of small GTPases of the Ras-dva family allowed us to determine the C-terminal prenylation motif, which could be responsible for the membrane localization of these proteins. We demonstrated using the in vivo EGFP-tracing that the Ras-dva small GTPases from the Xenopus laevis embryo-cells and NIH-3T3 fibroblasts are localized on both plasma membranes and endomembranes (the endoplasmic reticulum, the Golgi apparatus, and vesicles). At the same time, the replacement of Cys residue, the SH group of which must be theoretically farnesylated, in the C-terminal prenylation motif of the Ras-dva small GTPase by the Ser residue prevented the membrane localization of the protein. These results indicate that the C-terminal prenylation site is critical for the membrane localization of small Ras-dva GTPases.     

山茶CjMYB1基因的克隆及表达分析

该研究通过序列比对分析,以野生红山茶和不同花色品种山茶为材料,采用PCR方法克隆CjMYB1基因,并通过生物信息学和表达分析对其进行初步研究,为深入研究山茶CjMYB1基因在花色形成和花发育过程的调控机理奠定理论基础。结果表明：(1)成功克隆获得山茶CjMYB1基因(GenBank登录号为OL347930),其开放阅读框长为879 bp,编码292个氨基酸,相对分子质量为33.17 kD;CjMYB1基因属于R2R3-MYB转录因子,且与拟南芥MYB基因家族的第7亚组处于同一分支。(2)荧光定量PCR分析发现,山茶CjMYB1基因在野生红山茶花芽中表达量最高,在萼片、花瓣、雄蕊和心皮中都有较高的表达量,推测其在山茶花器官发育中发挥着重要作用;在红色山茶品种中表达量较高,而在粉色、淡黄色、白色山茶品种中表达量较低,说明CjMYB1基因可能在红色山茶品种的花色苷合成途径中起到了关键作用。(3)亚细胞定位实验表明,CjMYB1蛋白定位在细胞核。