A simple method with liquid chromatography/tandem mass spectrometry for the determination of the six trichothecene mycotoxins in rice medium

A selective and speedy LC-MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC-MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76-106% with a spiked level at 1-6 microg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

Cutting Edge: Plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.     

Expression of a truncated form of the endoplasmic reticulum chaperone protein, σ1 receptor, promotes mitochondrial energy depletion and apoptosis

The σ1 receptor (σ(1)R) regulates endoplasmic reticulum (ER)/mitochondrial interorganellar Ca(2+) mobilization through the inositol 1,4,5-trisphosphate receptor (IP(3)R). Here, we observed that expression of a novel splice variant of σ(1)R, termed short form σ(1)R (σ(1)SR), has a detrimental effect on mitochondrial energy production and cell survival. σ(1)SR mRNA lacks 47 ribonucleotides encoding exon 2, resulting in a frameshift and formation of a truncated receptor. σ(1)SR localizes primarily in the ER at perinuclear regions and forms a complex with σ(1)R but not with IP(3)R in the mitochondrion-associated ER membrane. Overexpression of both σ(1)R and the truncated isoform promotes mitochondrial elongation with increased ER mitochondrial contact surface. σ(1)R overexpression increases the efficiency of mitochondrial Ca(2+) uptake in response to IP(3)R-driven stimuli, whereas σ(1)SR overexpression reduces it. Most importantly, σ(1)R promotes ATP production via increased mitochondrial Ca(2+) uptake, promoting cell survival in the presence of ER stress. By contrast, σ(1)SR suppresses ATP production following ER stress, enhancing cell death. Taken together, the newly identified σ(1)SR isoform interferes with σ(1)R function relevant to mitochondrial energy production under ER stress conditions, promoting cellular apoptosis.     

Rapid detection of blaIMP-6 by amplification refractory mutation system

Klebsiella pneumoniae resistant to almost all ß-lactams except imipenem designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. All ISMRK carries bla_IMP-6 which differs from bla_IMP-1 by only a single nucleotide at position 640. We devised a rapid detection system of bla_IMP-6 by using ARMS PCR.     

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca²⁺]_i in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca²⁺]_i in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca²⁺]_i in response to MG-132. MDL-28170 induced neither an increase in [Ca²⁺]_i in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.     

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.

Methods

We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ₁R^E102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.

Results

σ₁R^E102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ₁R suppressed ER stress-induced mitochondrial injury, whereas σ₁R^E102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ₁R^E102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca^2 + transporter inhibitor Ru360 mimicked the effects of σ₁R^E102Q overexpression, indicating that aberrant σ₁R-mediated mitochondrial Ca^2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ₁R^E102Q overexpression.

Conclusions

Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.

General significance

ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.     

Asymmetric Dimeric Structure of Ferredoxin-NAD(P) Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP

Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) catalyzes the reduction of NAD(P)⁺ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.