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- 2019
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Tidskriftsartikel (refereegranskat)
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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Duan, Jianxin, et al.
(författare)
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Understanding the Molecular Activity of Alkaline Sphingomyelinase (NPP7) by Computer Modeling
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:42, s. 9096-9105
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Tidskriftsartikel (refereegranskat)abstract
- The enzymes in the nucleotide pyrophosphatase/phosphodiesterase (NPP) family have various substrates such as nucleotides, phospholipids, and sphingolipids. The substrate specificity in relation to their structures is largely unknown because no mammalian NPP complex has been crystallized. NPP7, also called alkaline sphingomyelinase (alk-SMase), is a NPP family member that may have important implications in carcinogenesis and cholesterol absorption. The sequence of NPP7 is 36% similar to that of the closest NPP member, but NPP7 has no activity against nucleotides. In this work, we predict the three-dimensional structure of NPP7 by homology modeling using a recently crystallized NPP from bacteria. Using the model, we studied the substrate specificity of the enzyme by docking. The model generated explains the functional changes in previous mutagenesis studies and rationalizes the structural basis for the lack of activity toward nucleotides. An effort to shift the substrate specificity from sphingomyelin (SM) to nucleotide was not successful but revealed a site-directed mutation that increased activity toward SM. In conclusion, this is the first study to predict the structure of a mammalian NPP and its substrate specificity by molecular modeling. The information may be helpful in understanding the functional differences of NPP members.
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- Sun, Xiao-dong, et al.
(författare)
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Implementing a novel capture and ligation probe-PCR method in mass screen and treatment to support malaria elimination efforts in the China-Myanmar border region
- 2023
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Ingår i: Malaria Journal. - : BioMed Central (BMC). - 1475-2875. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundMass screening and treatment (MSAT) for malaria elimination lacks an ideal diagnostic tool to allow sensitive and affordable test of the target population in the field. This study evaluated whether Capture and Ligation Probe-PCR (CLIP-PCR) could be used in a field MSAT in Laiza City, Myanmar.MethodsOn day 0, two dried blood spots were collected from each participant. On day 1, all samples were screened for Plasmodium in a 20 m(2) laboratory with workbench, a biosafety cabinet, a refrigerator, a benchtop shaking incubator and a qPCR machine, by four technicians using CLIP-PCR with sample pooling, at a health clinic of the Chinese bordering town of Nabang. On day 2, all positives were followed up and treated.ResultsOf 15,038 persons (65% of the total population) screened, 204 (1.36%) were CLIP-PCR positives. Among them, 188, 14, and 2 were infected with Plasmodium vivax, Plasmodium falciparum, and P. vivax/P. falciparum mix, respectively. The testing capacity was 538 persons/day, with a cost of US$0.92 /person. The proportion of submicroscopic infection was 64.7%. All positive individuals received treatment within 72 h after blood collection.ConclusionUsing CLIP-PCR in MSAT in low transmission settings can support the malaria elimination efforts in the China-Myanmar border region.
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- Alyamani, Manar, et al.
(författare)
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Alkaline sphingomyelinase (NPP7) impacts the homeostasis of intestinal T lymphocyte populations
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background and aim: Alkaline sphingomyelinase (NPP7) is expressed by intestinal epithelial cells and is crucial for the digestion of dietary sphingomyelin. NPP7 also inactivates proinflammatory mediators including platelet-activating factor and lysophosphatidylcholine. The aim of this study was to examine a potential role for NPP7 in the homeostasis of the intestinal immune system. Methods: We quantified the numbers of B-lymphocytes, plasma cells, T-lymphocytes including regulatory T-lymphocytes (Tregs), natural killer cells, dendritic cells, macrophages, and neutrophils, in the small and large intestines, the mesenteric lymph nodes and the spleens of heterozygous and homozygous NPP7 knockout (KO) and wildtype (WT) mice. Tissues were examined by immunohistochemistry and stainings quantified using computerized image analysis. Results: The numbers of both small and large intestinal CD3ε+, CD4+, and CD8α+ T-lymphocytes were significantly higher in NPP7 KO compared to WT mice (with a dose-response relationship in the large intestine), whereas Treg numbers were unchanged, and dendritic cell numbers reduced. In contrast, the numbers of CD3ε+ and CD4+ T-lymphocytes in mesenteric lymph nodes were significantly reduced in NPP7 KO mice, while no differences were observed in spleens. The numbers of B-lymphocytes, plasma cells, natural killer cells, macrophages, and neutrophils were similar between genotypes. Conclusion: NPP7 contributes to the regulation of dendritic cell and T-lymphocyte numbers in mesenteric lymph nodes and both the small and large intestines, thus playing a role in the homeostasis of gut immunity. Although it is likely that the downstream effects of NPP7 activity involve the sphingomyelin metabolites ceramide and spingosine-1-phosphate, the exact mechanisms behind this regulatory function of NPP7 need to be addressed in future studies.
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- Andersson, David, et al.
(författare)
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Expression of Alkaline Sphingomyelinase in Yeast Cells and Anti-inflammatory Effects of the Expressed Enzyme in a Rat Colitis Model.
- 2009
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Ingår i: Digestive Diseases and Sciences. - : Springer Science and Business Media LLC. - 1573-2568 .- 0163-2116. ; 2008:Nov 7, s. 1440-1448
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline sphingomyelinase (Alk-SMase) is a key enzyme in the intestinal tract for digestion of dietary sphingomyelin (SM), which generates lipid messengers with cell-cycle regulating effects. The enzyme is significantly decreased in ulcerative colitis and colon cancer. Based on this information, we wanted to investigate whether the enzyme had preventive effects against murine colitis. We report herein a method to express a biologically active Alk-SMase from Pichia pastoris yeast cells. By using the expressed enzyme to treat a rat colitis model induced by dextran sulfate sodium, we found that intrarectal instillation of Alk-SMase once daily for 1 week significantly reduced the inflammation score and protected the colonic epithelium from inflammatory destruction. We found a tendency for decreased tumor necrosis factor (TNF)-alpha expression in the Alk-SMase-treated group. This study, for the first time, provides a method to produce the enzyme and shows the potential applicability of the enzyme in the treatment of inflammatory bowel diseases.
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| 10. |
- Andersson, David, et al.
(författare)
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Ursolic acid and other pentacyclic triterpenoids stimulate intestinal alkaline sphingomyelinase in vitro
- 2006
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Ingår i: European Journal of Lipid Science and Technology. - : Wiley. - 1438-7697 .- 1438-9312. ; 108:2, s. 103-108
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Alkaline sphingomyelinase (alk-SMase) is an enzyme that hydrolyses sphingomyelin in a bile salt-dependent manner in the gastrointestinal tract, and has been proposed as an inhibitor of colon carcinogenesis. Ursolic acid (UA) is a plant-derived pentacyclic triterpenoid that has been shown to have anti-proliferative and apoptotic effects on HT29 human colon adenocarcinoma cells, with activation of alk-SMase as an early event. The aim of this study was to study the in vitro effects of UA and its analogues on the activity of purified rat intestinal alk-SMase. Methods: Rat intestinal alk-SMase activity was determined after incubation with UA in the presence and absence of taurocholate (TC). The effect was compared with boswellic acids, another group of pentacyclic triterpenoids. Results: UA enhanced the activity of rat intestinal alk-SMase in a dose-dependent manner, without a similar effect on bacterial neutral SMase. Four types of boswellic acid also increased the enzyme activity, with the effect of acetyl-keto-beta-boswellic acid being most potent. Activation of alk-SMase by TC; at a low concentration (0.4 mM), but not at a high concentration, was enhanced by UA. Conclusions: Ursolic acid and four types of boswellic acid, all pentacyclic triterpenoids, have a stimulatory effect on the activity of intestinal alk-SMase.
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