Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay

The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl- /-). Although c-mpl-/- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of 125I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl- /- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize 125I-TPO, whereas PRP from c-mpl-/- mice lacked this ability. Analysis of 125I-TPO binding to normal PRP indicated that binding was specific and saturable, with an approximate affinity of 560 pmol/L and 220 receptors per platelet. PRP from normal mice was also able to degrade 125I-TPO into lower molecular weight fragments. After the intravenous injections, c-mpl-/- mice cleared a dose of 125I-TPO at a much slower rate than did normal mice. Injection of washed platelets from normal mice into c-mpl-/- mice resulted in a dramatic, but transient, decrease in plasma TPO levels. These data provide evidence that platelets regulate plasma TPO levels via binding to the c-mpl receptor on circulating platelets.

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Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel FluorescenceIn SituHybridization Assay

Applied and Environmental Microbiology ◽

10.1128/aem.03457-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1114-1125 ◽

Cited By ~ 10

Author(s):

Theresa K. Hattenrath-Lehmann ◽

Yu Zhen ◽

Ryan B. Wallace ◽

Ying-Zhong Tang ◽

Christopher J. Gobler

Keyword(s):

Harmful Algal Blooms ◽

Algal Blooms ◽

Large Subunit ◽

Cross Reactivity ◽

Epifluorescence Microscopy ◽

Lsu Rdna ◽

Cochlodinium Polykrikoides ◽

Content Type ◽

Ecosystem Studies ◽

Resting Cysts

ABSTRACTCochlodinium polykrikoidesis a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated thatCochlodiniumforms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution ofC. polykrikoidescysts in coastal ecosystems. Here, we report on the development of a fluorescencein situhybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) ofC. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction withC. polykrikoidesand minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates ofC. polykrikoidesbut not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and mapC. polykrikoidescysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities ofC. polykrikoidescysts (>100 cm−3) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification ofC. polykrikoidesrDNA. This study mappedC. polykrikoidescysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.

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The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2010.1588 ◽

2010 ◽

Vol 1 (1) ◽

pp. 2

Author(s):

Sandra Ebert ◽

Line Breumlund ◽

Roland Nau ◽

Uwe Michel

Keyword(s):

Internal Standard ◽

Human Neuroblastoma Cell ◽

Protection Assay ◽

Human Neuroblastoma ◽

Rnase Protection ◽

S1 Nuclease ◽

Total Rna ◽

16S Rna ◽

Nuclease Protection Assay ◽

Mitochondrial Transcripts

The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochondrial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y) revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

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