Inter-laboratory study on simultaneous quantification of ten trichothecenes in feed.

An inter-laboratory study was performed in eight laboratories to evaluate the simultaneous quantification method for HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS), neosolaniol (NES), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deoxynivalenol (DON), deoxynivalenol-3-glucoside (D3G), nivalenol (NIV), and fusarenon-X (FUS-X) in feed. The mycotoxins in the samples were extracted with hydrous acetonitrile, purified using a multifunctional column (InertSep® VRA-3) and a phospholipid removal column (Hybrid SPE®-Phospholipid), and then quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionisation mode. The mean recovery, repeatability, reproducibility, and Horwitz ratio from the inter-laboratory validation study were 99.8-109%, 3.1-9.8%, 4.3-9.8%, and 0.19-0.45, respectively, for type A trichothecenes (HT-2, T-2, DAS, and NES). Those values for type B trichothecenes (3-AcDON, 15-AcDON, DON, NIV, and FUS-X) were 89.9-116%, 3.4-9.1%, 5.6-14%, and 0.25-0.70, and the values for modified mycotoxin (D3G) were 78.2-96.7%, 3.5-6.4%, and 13-22%, respectively.

Multiple system atrophy variant with severe hippocampal pathology.

The striatonigral and olivopontocerebellar systems are known to be vulnerable in multiple system atrophy (MSA), showing neuronal loss, astrogliosis, and alpha-synuclein-immunoreactive inclusions. MSA patients who displayed abundant neuronal cytoplasmic inclusions (NCIs) in the regions other than the striatonigral or olivopontocerebellar system have occasionally been diagnosed with variants of MSA. In this study, we report clinical and pathologic findings of MSA patients characterized by prominent pathologic involvement of the hippocampus. We assessed 146 consecutively autopsied MSA patients. Semi-quantitative analysis of anti-alpha-synuclein immunohistochemistry revealed that 12 of 146 patients (8.2%) had severe NCIs in two or more of the following areas: the hippocampal granule cells, cornu ammonis areas, parahippocampal gyrus, and amygdala. In contrast, the remaining 134 patients did not show severe NCIs in any of these regions. Patients with severe hippocampal involvement showed a higher representation of women (nine women/three men; Fisher's exact test, p = 0.0324), longer disease duration (13.1 ± 5.9 years; Mann-Whitney U-test, p = 0.000157), higher prevalence of cognitive impairment (four patients; Fisher's exact test, p = 0.0222), and lower brain weight (1070.3 ± 168.6 g; Mann-Whitney U-test, p = 0.00911) than other patients. The hippocampal granule cells and cornu ammonis area 1/subiculum almost always showed severe NCIs. The NCIs appeared to be ring-shaped or neurofibrillary tangle-like, fibrous configurations. Three of 12 patients also had dense, round-shaped NCIs that were morphologically similar to pick bodies. The patients with Pick body-like inclusions showed more severe atrophy of the medial temporal lobes and broader spreading of NCIs than those without. Immunohistochemistry for hyperphosphorylated tau and phosphorylated TDP-43 revealed minimal aggregations in the hippocampus of the hippocampal MSA patients. Our observations suggest a pathological variant of MSA that is characterized by severe involvement of hippocampal neurons. This phenotype may reinforce the importance of neuronal alpha-synucleinopathy in the pathogenesis of MSA.

[Determination of Inorganic Arsenic for Pet Food by LC-ICP-MS].

We have developed a quantitative determination method of the concentration of inorganic arsenic in pet foods using a liquid chromatograph-inductively coupled plasma-mass spectrometer (LC-ICP-MS). After adding 2 w/v% TMAH solution to a sample, inorganic arsenic was extracted by heating and the extract was collected by water. The pH of the solution was adjusted, and injected into a LC-ICP-MS to determine the concentration of inorganic arsenic. LC separation was carried out on an ODS column with 10 mmol/L sodium 1-butanesulfonate, 4 mmol/L malonic acid, 4 mmol/L TMAH and 0.05% methanol solution as a mobile phase. A collaborative study was conducted by nine laboratories using dry and wet-type pet foods, formed jerky, dried jerky and biscuit. Dry-type pet food and dried jerky was added with 2 mg/kg of As (III). Wet-type pet food was added with 0.5 mg/kg of As (III). Formed jerky was added with 1 mg/kg of As (III). Biscuit was added with 0.2 mg/kg of As (III). The mean recoveries, repeatabilities and reproducibilities in the form of relative standard deviation (RSDr and RSDR), and HorRat, were 95.4% to 98.3%, less than 2.9%, less than 9.1%, and 0.22 to 0.51, respectively.

Development of an In Vitro Chloroplast Splicing System: Sequences Required for Correct pre-mRNA Splicing.

Chloroplast genomes in land plants include approximately 20 intron-containing genes. Most of the introns are similar to the group II introns found in fungi, algae and some bacteria, but no self-splicing has been reported. To analyze splicing reactions in chloroplasts, we developed a tobacco (Nicotiana tabacum) chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor messenger RNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is not necessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 and 2) complementary to two intron-binding sites (IBS1 and 2) in the 3' end of 5' exons, which are involved in 5' splice-site selection. Using our in vitro system and atpF pre-mRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1-IBS1 pairing is essential, while EBS2-IBS2 pairing is important but not crucial for splicing. The first 3' exon nucleotide determines the 3' splice sites of self-splicing introns. However, mutations to this nucleotide in atpF pre-mRNA did not affect splicing. This result suggests that the mechanism underlying chloroplast pre-mRNA splicing differs partly from that mediating the self-splicing of group II introns.

Development of a simultaneous quantification method for ten trichothecenes including deoxynivalenol-3-glucoside in feed.

An analytical method for the simultaneous quantitation of ten trichothecenes of type A (HT-2 toxin, T-2 toxin, diacetoxyscirpenol, and neosolaniol) and type B (3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, nivalenol, and fusarenon-X) in feed has been developed using liquid chromatography with tandem mass spectrometry. Mycotoxins extracted twice from samples using aqueous acetonitrile were purified using a multifunctional clean-up column, followed by a phospholipid removal column. Trichothecenes were analysed using liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry. The extraction efficiency of the mycotoxins and the repeatability of some were improved by repeated extractions. Ionization enhancement (signal enhancement) of some mycotoxins was improved by using the phospholipid removal column at the clean-up step. Spike and recovery tests of trichothecenes were conducted on maize, barley, soybean meal, rapeseed meal, and formula feeds (for starting broiler chicks, suckling pigs, and beef cattle). The mean recovery values were 70.6-119% with relative standard deviations < 17%. The limit of quantification and the limit of detection of our method were 20 and 6 µg/kg, respectively, for 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol; 10 and 3 µg/kg, respectively, for T-2 toxin, deoxynivalenol, and fusarenon-X; and 5 and 2 µg/kg, respectively, for nivalenol and the remaining mycotoxins.

Sterigmatocystin and aflatoxin B1 contamination of corn, soybean meal, and formula feed in Japan.

Sterigmatocystin (STC) and aflatoxin B1 (AFB1) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 µg/kg, and 1.2 µg/kg for corn; 14%, 1.1 µg/kg, and 0.63 µg/kg for soybean meal; and 43%, 9.1 µg/kg, and 0.97 µg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB1 were respectively 46%, 24 µg/kg, and 3.9 µg/kg for corn; 30%, 6.7 µg/kg, and 1.1 µg/kg for soybean meal; and 47%, 20 µg/kg, and 1.6 µg/kg for formula feed. A weak negative correlation between the STC and AFB1 concentrations was observed: there was a high concentration of AFB1 in samples that contained a lower concentration of STC and vice versa. Spearman's rank correlation coefficient showed a weak negative correlation of - 0.30 (p < 0.001, n = 128) for corn and - 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 µg/kg) and in corn (1.2 µg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER­deficient disorders.

Subcutaneous phaeohyphomycosis caused by Exophiala jeanselmei in collagen disease patient.

Phaeohyphomycosis is a rare fungal infection that is more commonly associated with compromised patients. We present herein an 81-year-old man with collagen disease and chronic interstitial pneumonia who developed subcutaneous phaeohyphomycosis caused by Exophiala jeanselmei. The main pathogen of phaeohyphomycosis had been considered to be E. jeanselmei complex. This has recently been divided into several species by using a molecular technique. The main pathogen of phaeohyphomycosis is Exophiala xenobiotica, and E. jeanselmei is rather a rare pathogen of this disease. Although p.o. itraconazole and terbinafine administration was not effective for this patient, these antifungal agents were used for preventing systemic dissemination in this immunocompromised host.

Lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal phylogenetic diversity in heat shock protein 70 sequences.

We previously found that mouse inducible Hsp72 bound more extensively to lymphoblast-like lymphoid neoplastic P388D1 cells than to RAW264.7 monocyte-macrophages. In the present study, we analyzed the characteristics of the binding to P388D1 cells of recombinant HSP70 derived from different species. Recombinant mouse inducible-type Hsp72 bound extensively to P388D1 cells in a saturable manner, but not to P815 mastocytoma or EL4 thymoma. Spinach Hsc70-1, highly homologous with mouse Hsp72 in the C-terminal region, also bound to P388D1 cells. In contrast, significant binding was not observed for bacterial DnaK derived from Lactobacillus acidophilus and Escherichia coli which have relatively little homology in this region. Analyses of surface antigens showed that B220, and CD19, but not CD91, LOX-1, and CD40, the HSP70 receptors reported so far, were present on P388D1 cells, suggesting a B-cell lineage for this cell line. A similar discrimination of the diversity between mouse Hsp72 and bacterial DnaK occurred for CD19(+) B cells derived from mouse spleen, Peyer's patches, and mesenteric lymph nodes. The binding of HSP70 to P388D1 cells was partially, but significantly, antagonized by fucoidan and maleylated BSA, implying a few types of scavenger receptors to be responsible for the binding of HSP70 to this cell line. Furthermore, photo-affinity labeling revealed several membrane protein candidates larger than 110kDa to be involved in the recognition of HSP70 molecules. Using a NF-κB-luciferase reporter assay, we found that exogenous HSP70 did not stimulate NF-κB-dependent signal transduction in P388D1 cells. It thus follows that lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal sequences of the HSP70 family. The present results indicate that several types of cells in the innate immune system may distinguish among the phylogenetically specific signals in protein molecules.

Subcutaneous phaeohyphomycosis caused by Exophiala xenobiotica in a non-Hodgkin lymphoma patient.

Phaeohyphomycosis is a rare fungal infection that is more commonly associated with immunocompromised patients. We present a case in which a 77-year-old woman with non-Hodgkin lymphoma developed subcutaneous phaeohyphomycosis caused by Exophiala xenobiotica. E. xenobiotica is a dematiaceous hyphomycete that was recently identified as a segregant of the E. jeanselmei complex. The patient was successfully treated with local excision of the lesions and post-surgical oral itraconazole. The latter was administered with the aim of preventing systemic dissemination in this immunocompromised patient.

Paroxysmal kinesigenic choreoathetosis (PKC): confirmation of linkage to 16p11-q21, but unsuccessful detection of mutations among 157 genes at the PKC-critical region in seven PKC families.

Paroxysmal kinesigenic choreoathetosis (PKC) is a paroxysmal movement disorder of unknown cause. Although the PKC-critical region (PKCCR) has been assigned to the pericentromeric region of chromosome 16 by several studies of families from various ethnic backgrounds, the causative gene has not yet been identified. In the present study, we performed linkage and haplotype analysis in four new families with PKC, as well as an intensive polymerase chain reaction (PCR) based mutation analysis in seven families for a total of 1,563 exons from 157 genes mapped around the PKCCR. Consequently, the linkage/haplotype analysis revealed that PKC was assigned to a 24-cM segment between D16S3131 and D16S408, the result confirming the previously defined PKCCR, but being unable to narrow it down. Although the mutation analysis of the 157 genes was unsuccessful at identifying any mutations that were shared by patients from the seven families, two nonsynonymous substitutions, i.e., 6186C>A in exon 3 of SCNN1G and 45842A>G in exon 29 of ITGAL, which were segregated with the disease in Families C and F, respectively, were not observed in more than 400 normal controls. Thus, one of the two genes, SCNN1G and ITGAL, could be causative for PKC, but we were not able to find any other mutations that explain the PKC phenotype.

Mouse CD40-transfected cell lines cannot exhibit the binding and RANTES-stimulating activity of exogenous heat shock protein 70.

Here we demonstrate the inducible mouse Hsp72 binds markedly to lymphoid neoplastic macrophage-like P388D1 cells. To examine whether mouse CD40 can play a role in signaling exogenously administered HSP70 in a fashion similar to that of human CD40, we established mouse CD40-transfectants of both human 293 cells and murine-pro-B cell line Ba/F3. A small portion of mouse CD40 expressed on 293-derived transfectants was the mature form with a signal-transducible C-terminal domain, whereas a majority of expressed antigen showed the molecular size smaller than we expect. Flow cytometry showed that mouse Hsp72, but neither its deletion variants nor the related Escherichia coli DnaK, bound to the 293-derived transfectants regardless of CD40 expression. CD40 molecules expressed on the transfectants showed the binding of soluble form of CD40L but this binding was not inhibited by excess amount of HSP70. CD40L, but not any HSP70 recombinant proteins, stimulated the production of chemokine RANTES in the transfectants. Furthermore, no RANTES production was induced by HSP70-RCMLA complex in the transfectants, although it binds to 293-derived cells in a CD40-independent manner. No interaction between mouse CD40 and HSP70 recombinant proteins was detected by using the Ba/F3-derived transfectants that express the mature form of mouse CD40. The present results imply that mouse CD40 expressed on the transfectants differs from its human homolog in the binding of exogenously administered HSP70.

BAC array CGH reveals genomic aberrations in idiopathic mental retardation.

Array using 2,173 BAC clones covering the whole human genome has been constructed. All clones spotted were confirmed to show a unique signal at the predicted chromosomal location by FISH analysis in our laboratory. A total of 30 individuals with idiopathic mental retardation (MR) were analyzed by comparative genomic hybridization using this array. Three deletions, one duplication, and one unbalanced translocation could be detected in five patients, which are likely to contribute to MR. The constructed array was shown to be an efficient tool for the detection of pathogenic genomic rearrangements in MR patients as well as copy number polymorphisms (CPNs).

A SNP in the ABCC11 gene is the determinant of human earwax type.

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G --> A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G --> A SNP is the first example of DNA polymorphism determining a visible genetic trait.

Biotinylation of heat shock protein 70 induces RANTES production in HEK293 cells in a CD40-independent pathway.

Biotinylated proteins and peptides have been used as popular ligands for characterization of cell surface receptors by a variety of methods including flow cytometry. The number and the location of biotin moieties incorporated could alter the structural and physicochemical properties of ligands, although biotin is thought to be such a small molecule (244Da) that it is capable of being conjugated to most proteins without affecting their activity. Here, we demonstrate that the biotinylated HSP70 molecule via primary amines bound to epithelium-like HEK 293 cells in a saturable manner whereas the unlabeled counterparts of HSP70 other than mouse Hsp72 do not. This binding was not competed by either HSP70 or the biotin entity itself. Interestingly, the biotinylated HSP70 also elicited the production of CC-chemokine RANTES independent of CD40 signaling. This response occurred regardless of sequence diversity of HSP70 derived from different species, and neither the biotinylated ovalbumin nor the unlabeled HSP70 cross-linked with a biotinylated protein stimulated a significant level of RANTES production which was induced by biotinylated HSP70 itself. Our findings suggest that modification of HSP70 such as biotinylation may function as a biological alarm signal in the innate immune system.

Eosinophil infiltration in three patients with generalized atrophic benign epidermolysis bullosa from a Japanese family: molecular genetic and immunohistochemical studies.

Generalized atrophic benign epidermolysis bullosa (GABEB), a subtype of epidermolysis bullosa (EB), is an autosomal recessive skin disease characterized by derm-epidermal separation leading to skin fragility and atrophy and other associated abnormalities. Although a few reports demonstrated that eosinophils are infiltrating beneath bullas in infants with some types of EB, no such condition in adult GABEB patients has been known. Here we report on three adult patients with GABEB from a Japanese family, whose bullous skin lesions showed massive eosinophil infiltration. One of the three patients showed amyloid deposition at the intestine, kidney, and skin. Linkage analysis revealed that GABEB in the family was linked to COL17A1 with a maximum LOD score of 3.08. Mutation analysis identified in the three patients a homozygous insertional mutation, 209-210insCA, in exon 5 of COL17A1. The expression of mutated COL17A1 was confirmed by semiquantitative RT-PCR, but no signals for truncated COL17A1 protein were detected by the immunohistochemical studies using antibodies against BP180. Furthermore, no autoantibody against the mutant protein was detected by western blot analysis. It is thus less likely that autoantibody and/or a local immune reaction in their skin has a primary role in eosinophil infiltration in these patients.