Inhibition of Amyloid Precursor Protein Processing Enhances Gemcitabine-mediated Cytotoxicity in Pancreatic Cancer Cells

Pancreatic adenocarcinoma or pancreatic cancer is often diagnosed at a very late stage at which point treatment options are minimal. Current chemotherapeutic interventions prolong survival marginally, thereby emphasizing the acute need for better treatment options to effectively manage this disease. Studies from different laboratories have shown that the Alzheimer disease-associated amyloid precursor protein (APP) is overexpressed in various cancers but its significance is not known. Here we sought to determine the role of APP in pancreatic cancer cell survival and proliferation. Our results show that pancreatic cancer cells secrete high levels of sAPPα, the α-secretase cleaved ectodomain fragment of APP, as compared with normal non-cancerous cells. Treatment of cells with batimastat or GI254023X, inhibitors of the α-secretase ADAM10, prevented sAPPα generation and reduced cell survival. Additionally, inhibition of sAPPα significantly reduced anchorage independent growth of the cancer cells. The effect of batimastat on cell survival and colony formation was enhanced when sAPPα downregulation was combined with gemcitabine treatment. Moreover, treatment of batimastat-treated cells with recombinant sAPPα reversed the inhibitory effect of the drug thereby indicating that sAPPα can indeed induce proliferation of cancer cells. Down-regulation of APP and ADAM10 brought about similar results, as did batimastat treatment, thereby confirming that APP processing is important for growth and proliferation of these cells. These results suggest that inhibition of sAPPα generation might enhance the effectiveness of the existing chemotherapeutic regimen for a better outcome.     

Identification of a Region within the Placental Alkaline Phosphatase mRNA That Mediates p180-dependent Targeting to the Endoplasmic Reticulum

In both eukaryotic and prokaryotic cells, it has been recently established that mRNAs encoding secreted and membrane proteins can be localized to the surface of membranes via both translation-dependent and RNA element-mediated mechanisms. Previously, we showed that the placental alkaline phosphatase (ALPP) mRNA can be localized to the ER membrane independently of translation, and this localization is mediated by p180, an mRNA receptor present in the ER. In this article, we aimed to identify the cis-acting RNA element in ALPP. Using chimera constructs containing fragments of the ALPP mRNA, we demonstrate that the ER-localizing RNA element is present within the 3′ end of the open reading frame and codes for a transmembrane domain. In addition, we show that this region requires p180 for efficient ER anchoring. Taken together, we provide the first insight into the nature of cis-acting ER-localizing RNA elements responsible for localizing mRNAs on the ER in mammalian cells.     

Ribosomal protein S3 is secreted as a homodimer in cancer cells

Protein secretion is a general phenomenon by which cells communicate with the extracellular environment. Secretory proteins, including hormones, enzymes, toxins, and antimicrobial peptides have various functions in extracellular environments. Here, we determined that ribosomal protein S3 (rpS3) is homodimerized and secreted in several cancer cell lines such as HT1080 (human fibrosarcoma) and MPC11 (mouse plasmacytoma). Moreover, we found that the secreted rpS3 protein increased in doxorubicin-resistant MPC11 cells compared to that in MPC11 cells. In addition, we also detected that the level of secreted rpS3 increased in more malignant cells, which were established with continuous exposure of cigarette smoke condensate. These findings suggest that the secreted rpS3 protein is an indicator of malignant tumors.     

2 型糖尿病患者胰高血糖素及胰岛素水平检测及临床意义

目的:探讨2型糖尿病患者经标准馒头餐试验后胰高血糖素、胰岛素水平变化及其临床意义。方法:选取我院2014年3月至2015年3月收治的80例2型糖尿病患者为试验组,均接受标准馒头餐试验,对其餐前及餐后0.5 h、1 h、2 h胰高血糖素及胰岛素水平进行检测,并与同期80例血糖无异常的健康对照组作比较。结果:健康对照组餐前及餐后胰高血糖素水平无明显变化(P0.05),而试验组餐前胰高血糖素水平明显高于健康对照组(P0.05),且餐后明显升高,在餐后1 h达到峰值,餐后0.5 h、1h、2 h均明显高于健康对照组(P0.01)。健康对照组餐后胰岛素水平明显升高,在餐后1 h达到峰值后开始降低,较餐前略高,但差异平无统计学意义(P0.05);试验组餐前胰岛素水平明显低于健康对照组(P0.05),且餐后水平升高幅度缓慢,餐后0.5 h、1 h、2h均明显低于健康对照组(P0.05);实验组胰岛素抵抗指数(HOMA-IR)明显高于健康对照组,经Pearson相关分析,试验组餐后0.5 h、1 h、2 h胰高血糖素水平与HOMA-IR均呈明显的正相关(r=0.273、0.335、0.368,P0.05)。结论:2型糖尿病患者血糖高表达与胰高血糖素、胰岛素水平密切相关,胰高血糖素可拮抗胰岛素,及时检测二者的水平对临床准确评估病情具有重要的意义。     

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.     

Somatostatin induces rapid contraction of neuroendocrine cells

The peptide hormone somatostatin, as well as the somatostatin analog octreotide, induces rapid morphological changes in neuroendocrine cells. The effect can be detected in less than 2 min: retraction fibers are formed, cells round up and cell-cell contacts are broken. Somatostatin-dependent cell contraction is inhibited by Y-27632, indicating that this effect is dependent on Rho kinase. In BON1 cells, the somatostatin-induced inhibition of forskolin-induced secretion of chromogranin A is not blocked by Y-27632. It is therefore concluded that the inhibitory effect of somatostatin in forskolin-stimulated cells is not dependent on cell contraction.     

Signal sequence recognition and protein targeting

Intracellular traffic is often controlled not by highways, but by handshakes and partner introductions within a cellular network. Recently determined structures suggest how signal sequences are recognized and how the GTP affinities of the signal recognition particle and its receptor are coupled to the targeting of ribosomes to translocational membrane pores. The structure of signal peptidase suggests how it releases functional proteins.     

Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator

HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC. HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37° C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites. However, the effect of removing both HlyB and TolC is not additive. On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC. This suggests that the presence of HlyB induces a structural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does. This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective. Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active haemolysin is limited to a brief period during mid to late exponential phase. In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion. Received: 10 July 1998 / Accepted: 19 October 1998     

Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp

We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S. albogriseolus which processes the pro-region. In the present study, we have used a chimeric pro-region consisting of S. mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of MTGase in C. glutamicum. As a result, secretion of MTGase using the chimeric pro-region is increased compared to that using the native pro-region.     

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.