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1.
PeerJ Comput Sci ; 9: e1620, 2023.
Article in English | MEDLINE | ID: mdl-37869462

ABSTRACT

Purpose: The purpose of this study is to compare two libraries dedicated to the Markov chain Monte Carlo method: pystan and numpyro. In the comparison, we mainly focused on the agreement of estimated latent parameters and the performance of sampling using the Markov chain Monte Carlo method in Bayesian item response theory (IRT). Materials and methods: Bayesian 1PL-IRT and 2PL-IRT were implemented with pystan and numpyro. Then, the Bayesian 1PL-IRT and 2PL-IRT were applied to two types of medical data obtained from a published article. The same prior distributions of latent parameters were used in both pystan and numpyro. Estimation results of latent parameters of 1PL-IRT and 2PL-IRT were compared between pystan and numpyro. Additionally, the computational cost of the Markov chain Monte Carlo method was compared between the two libraries. To evaluate the computational cost of IRT models, simulation data were generated from the medical data and numpyro. Results: For all the combinations of IRT types (1PL-IRT or 2PL-IRT) and medical data types, the mean and standard deviation of the estimated latent parameters were in good agreement between pystan and numpyro. In most cases, the sampling time using the Markov chain Monte Carlo method was shorter in numpyro than that in pystan. When the large-sized simulation data were used, numpyro with a graphics processing unit was useful for reducing the sampling time. Conclusion: Numpyro and pystan were useful for applying the Bayesian 1PL-IRT and 2PL-IRT. Our results show that the two libraries yielded similar estimation result and that regarding to sampling time, the fastest libraries differed based on the dataset size.

2.
Phytochemistry ; 89: 15-25, 2013 May.
Article in English | MEDLINE | ID: mdl-23453909

ABSTRACT

Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 µg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.


Subject(s)
Cucurbitaceae/metabolism , Lepidoptera/drug effects , Phloem/metabolism , Plant Lectins/genetics , Plant Lectins/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cucurbitaceae/physiology , DNA, Complementary/genetics , Escherichia coli/genetics , Gene Expression , Hemagglutination/drug effects , Lepidoptera/growth & development , Molecular Sequence Data , Plant Lectins/chemistry , Plant Lectins/isolation & purification , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
3.
Article in English | MEDLINE | ID: mdl-23267349

ABSTRACT

Neuropeptide W (NPW), which was first isolated from the porcine hypothalamus, exists in two forms, consisting of 23 (NPW23) or 30 (NPW30) amino acids. These neuropeptides bind to one of two NPW receptors, either NPBWR1 (otherwise known as GPR7) or NPBWR2 (GPR8), which belong to the G protein-coupled receptor family. GPR7 is expressed in the brain and peripheral organs of both humans and rodents, whereas GPR8 is not found in rodents. GPR7 mRNA in rodents is widely expressed in several hypothalamic regions, including the paraventricular, supraoptic, ventromedial, dorsomedial, suprachiasmatic, and arcuate nuclei. These observations suggest that GPR7 plays a crucial role in the modulation of neuroendocrine function. The intracerebroventricular infusion of NPW has been shown to suppress food intake and body weight and to increase both heat production and body temperature, suggesting that NPW functions as an endogenous catabolic signaling molecule. Here we summarize our current understanding of the distribution and function of NPW in the brain.

4.
Neuropeptides ; 46(6): 285-9, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23110814

ABSTRACT

Neuropeptide Y (NPY) is a 36-amino-acid neuropeptide that was first discovered in porcine brain extracts and later in the porcine intestine. It is widely distributed in both the central and peripheral nervous systems and exerts a powerful orexigenic effect. NPY-producing neuronal cell bodies are abundantly localized in the medial arcuate nucleus of the hypothalamus, this being a brain center that integrates signals for energy homeostasis. Accumulated evidence shows that hypothalamic neuropeptides such as ghrelin, orexin, melanin-concentrating hormone (MCH), galanin-like peptide (GALP) and proopiomelanocortin (POMC) are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. NPY also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. We summarize here current knowledge of the neuronal interactions between NPY and the different types of feeding-regulating peptide-containing neurons in the hypothalamus based on evidence at the immunohistochemicl level and with calcium imaging techniques.


Subject(s)
Arcuate Nucleus of Hypothalamus/physiology , Eating/physiology , Neurons/physiology , Neuropeptide Y/physiology , Animals , Galanin/physiology , Ghrelin/physiology , Humans , Hypothalamic Hormones/physiology , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/physiology , Leptin/physiology , Melanins/physiology , Neuropeptides/physiology , Orexins , Pituitary Hormones/physiology , Signal Transduction/physiology
5.
Methods Mol Biol ; 623: 197-209, 2010.
Article in English | MEDLINE | ID: mdl-20217553

ABSTRACT

We describe two efficient and inexpensive methods for reverse transfection with siRNA from a solid surface. One method involves localized reverse transfection from spots on a glass slide, which is mainly useful for making "transfection microarrays" (TMAs). The other involves reverse transfection in multiple wells of microtiter plates. Conditions for cell culture, preparation of reagents, and details of reverse transfection have been determined for several lines of cells, but we focus here on experiments with HeLa cells. In particular, we evaluated the efficiency of transfection, the cytotoxic effects of reverse transfection, and the efficiency of gene "knockdown" by transfection. We also performed phenotypic screening for a functional gene, during which cell viability was evaluated in terms of fluorescence from Calcein-AM. Our methods for reverse transfection with siRNA should be powerful tools that are useful for high-throughput analysis of functional genes.


Subject(s)
Transfection/methods , Gene Knockdown Techniques , HeLa Cells , Humans , RNA, Small Interfering/genetics
6.
J Biosci Bioeng ; 104(4): 329-33, 2007 Oct.
Article in English | MEDLINE | ID: mdl-18023808

ABSTRACT

We have developed an efficient and inexpensive method of reverse transfection from the solid phase to suppress genes with siRNA. The method enabled the realization of (i) a high efficiency of transfection; (ii) transfection of various types of cell; (iii) a high efficiency of gene knockdown by siRNA; (iv) a low toxicity to cells; and (v) a long-term stabilization (more than 210 d) of attached transfection mixture including siRNA in multiple wells. Although array-based reverse transfection has advantages in terms of miniaturization, the method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate. Our method of reverse transfection with siRNA in multiple wells is a powerful and high-throughput tool for the analysis of signaling pathways.


Subject(s)
Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , RNA, Small Interfering/genetics , Transfection/instrumentation , Transfection/methods
7.
Toxicon ; 48(8): 1002-10, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-16973201

ABSTRACT

Plancitoxin I, the major lethal factor from the crown-of-thorns starfish Acanthaster planci venom, is quite unique not only in exhibiting potent hepatotoxicity but also in sharing high sequence homology with mammalian deoxyribonulease II. In this study, morphological and biochemical changes in rat liver epithelial cells (TRL 1215 cells) treated with the toxin were examined to understand the mechanism by which plancitoxin I displays hepatotoxicity. AlamarBlue assay established that plancitoxin I is cytolethal to TRL 1215 cells. This cytolethalithy was ascribable to apoptotic cell death. Nuclear fragmentation evidenced by either Diff-Quick or Hoechst 33258 staining, DNA fragmentation by TUNEL assay and electrophoretic analysis on agarose gel and phosphatidylserine externalization by flow cytometric analysis of annexin V-FITC stained cells were all characteristics of apoptosis. The observed apoptosis was shown to be independent of the caspase 3 cascade that is generally accepted as the effector of the apoptotic process. Very interestingly, experiments using FITC-labeled plancitoxin I proved that the toxin can enter the nucleus of TRL 1215 cells. Our results suggested that plancitoxin I induces apoptosis of TRL 1215 cells through the following procedure: binding to a specific receptor in the cytoplasmic membrane, entering the cell, entering the nucleus and degrading DNA.


Subject(s)
Apoptosis/drug effects , Caspase 3/metabolism , Liver/drug effects , Marine Toxins/toxicity , Starfish/chemistry , Venoms/toxicity , Animals , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cells, Cultured , Epithelial Cells/drug effects , Liver/cytology , Marine Toxins/chemistry , Marine Toxins/metabolism , Protein Transport , Rats , Venoms/chemistry
8.
Article in English | MEDLINE | ID: mdl-16275035

ABSTRACT

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.


Subject(s)
Marine Toxins/genetics , Phospholipases A/genetics , Starfish/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calcium/metabolism , Cloning, Molecular , DNA, Complementary/genetics , Molecular Sequence Data , Peptide Fragments/metabolism , Phospholipases A2 , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid
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