Detection of an interaction between prion protein and neuregulin I-β1 by fluorescence resonance energy transfer analysis.

Cellular prion protein (PrP) copurifies with neuregulin type I-β1 (NRG I-β1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-β1. Here, the interaction of PrP with NRG I-β1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs).

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala.

A novel mucosal vaccine targeting Peyer's patch M cells induces protective antigen-specific IgA responses.

Mucosal vaccines can induce mucosal immunity, including antigen-specific secretory IgA production, to protect from invasion by pathogens and to neutralize toxins at mucosal surfaces. We established an effective antigen-delivering fusion protein, anti-GP2-SA, as a mucosal vaccine. The anti-GP2-SA consists of streptavidin (SA) fused to the antigen-binding fragment region from a mAb against glycoprotein 2 (GP2), an antigen-uptake receptor specifically expressed on M cells.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence.

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution.

Establishment of true niacin deficiency in quinolinic acid phosphoribosyltransferase knockout mice.

Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp.

Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.

Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.

Eosin-shadow method: a selective enhancement of light-microscopic visualization of pancreatic zymogen granules on hematoxylin-eosin sections.

We developed a novel method for enhancing light-microscopic visualization of pancreatic zymogen granules in a selective manner on hematoxylin and eosin-stained sections. By using an absorption filter that transmits light with wavelength from 510 to 550 nm, corresponding to the narrow absorption spectrum of eosin, only eosinophilic tissue and cellular components were remarkably highlighted as distinct shadows against lighter background consisting of basophilic components. Using a pair of mirror sections of the pancreas, immunocytochemistry with anti-amylase antibody confirmed that the shadows observed through the filter represented zymogen granules.

Crystal structures of sodium-bound annexin A4.

Annexin A4 (Anx4) possesses four repeat domains with one Ca(2+)-binding site (CBS) in each domain. In this study, we resolved two crystal structures of the Na(+)-bound form at high resolution (1.58 and 1.

Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E.

Phthalate esters enhance quinolinate production by inhibiting alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), a key enzyme of the tryptophan pathway.

Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism.

Identification and expression of alpha cDNA encoding human 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD): a key enzyme for the tryptophan-niacine pathway and quinolinate hypothesis.

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from 2-amino-3-carboxymuconate-6-semialdehyde (ACMS).

Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin.

Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells.

DNA-based identification of Brassica vegetable species for the juice industry.

Since kale (Brassica oleracea var. acephala), a cruciferous vegetable with a high level of vitamins and functional compounds beneficial to health and wellness, has become widely used in the juice industry, a precise method for quality control of vegetable species is necessary. We describe here a DNA-based identification method to distinguish kale from cabbage (Brassica oleracea var.

Phosphatidic acid production, required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells, is regulated by a wortmannin-sensitive process.

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells.

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin, reveals its resistance to proteinaceous trypsin inhibitors.

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown.

Identification and expression of a cDNA encoding human alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD). A key enzyme for the tryptophan-niacine pathway and "quinolinate hypothesis".

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS).

Expression of rat hepatic 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase is affected by a high protein diet and by streptozotocin-induced diabetes.

In the tryptophan-niacin conversion, 2-amino-3-carboxymuconate-6-semiardehyde decarboxylase (ACMSD; formerly termed picolinic carboxylase) is an important enzyme regulating the generation of quinolinate. In a series of experiments, we investigated alterations of ACMSD expression in rats by feeding a high protein diet and by inducing diabetes with streptozotocin (STZ). Male Sprague-Dawley rats (5-wk-old) were fed a diet containing 40% casein for 11 d, and hepatic ACMSD activity and mRNA expression were determined at intervals.

Cloning and characterization of ZAP36, an annexin-like, zymogen granule membrane associated protein, in exocrine pancreas.

ZAP36, a zymogen granule membrane associated protein with 36 kDa, was cloned from both canine and rat pancreas. ZAP36 is found to be a novel member of annexin IV, and showed an apical localization in exocrine pancreas and an ubiquitous expression in epithelial tissues. ZAP36 may be involved in exocytosis in apical regions of polarized cells.

Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase.

2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD; EC 4.1.1.