Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-ionization tandem mass spectrometry

Pyrroloquinoline quinone (PQQ) is believed to be an important factor for mammalian growth and development and has, therefore, been declared a vitamin by some researchers. However, this issue remains controversial, and from a nutritional viewpoint, accurate determination of PQQ levels in a variety of foods is very important. Here, we describe a simple, highly sensitive, and highly selective method for quantitative analysis of PQQ. Liquid foods or aqueous extracts of solid foods were analyzed using high-performance liquid chromatography (HPLC) combined with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS). (15)N-labeled PQQ was added to the samples as an internal standard. Quantitative analyses of PQQ were performed by multiple reaction monitoring (MRM) with LC/MS/MS. Free PQQ was detected in almost all food samples in the range 0.19-7.02 ng per g fresh weight (for solid foods) or per mL (liquid foods). This method will enable the rapid and simple determination of PQQ levels in many samples.     

Water status of hinoki cypress, Chamaecyparis obtusa, attacked by secondary woodboring insects after typhoon strike

We estimated the water status of six 31-year-old hinoki cypress (Chamaecyparis obtusa) trees that were struck by a large typhoon. Measurements were made of the trunk water-conduction area of xylem, leaf water potentials, and diurnal changes in trunk diameters. The secondary woodboring insects, Callidiellum rufipenne and Ovalisia vivata, on the trees were captured by the sticky-trap banding method. In trees on which these wood borers were captured, water conduction areas were reduced, leaf water potentials were lower, and trunk diameter changes (R) fluctuated during the day, suggesting that the trees had been damaged by the typhoon and were under water stress. The secondary woodboring insects attacked the water-stressed trees.     

Isolation of Listeria monocytogenes from the skin of slaughtered beef cattle

We attempted to isolate Listeria monocytogenes from skin, contents of large intestines and carcasses of cattle introduced to a slaughterhouse in order to identify source of contamination for this pathogen. Sixty skin samples, 60 samples of the contents of large intestines and 30 carcass samples were colleted in June, August and November 2003 for use in this study. Listeria spp. and L. monocytogenes were isolated from 30 (50%) and 3 (5%) of the cattle skin samples, respectively. However, no Listeria spp., including L. monocytogenes, were isolated from intestinal contents or carcasses. Seven isolates were obtained, of which five and two strains were serotypes 1/2a and 1/2b, respectively. Genetic analysis suggested that there was persistent inhabitation of the pathogen around the area investigated in this study.     

Damping-off of cabbage plug seedlings caused by Pythium ultimum var. ultimum in Japan

In October 2004, Pythium ultimum var. ultimum was isolated from rotten stems of cabbage plug seedlings in a commercial nursery in Mie Prefecture (Japan). The isolated fungus was then used to inoculate seedlings and subsequently reisolated from the seedlings with the damping-off disease, showing that P. ultimum var. ultimum is a new pathogen causing cabbage seedling disease.     

Multiplication of isolate R-5 of <Emphasis Type="Italic">Streptomyces galbus</Emphasis> on rhododendron leaves and its production of cell wall-degrading enzymes

 An endophytic actinomycete, isolate R-5 of Streptomyces galbus Frommer, that has promising potential as a biocontrol agent was originally isolated from field-grown rhododendron. In this study, the mode of entry of R-5 into leaves of tissue-cultured seedlings of rhododendron was investigated in connection with its production of cell wall-degrading enzymes. Light and scanning electron microscopy (SEM) revealed that R-5 grew on leaf surfaces and entered leaf tissues via stomata and that the internal mycelia grew out of stomata after colonization in host tissues. Micromanipulation at the SEM level demonstrated a prominent depression in the host surface at the interfaces with the mycelia, suggesting that such a depression could be caused by degradation of cell wall components by hydrolytic enzymes secreted from R-5 mycelia. In subsequent plate assays, R-5 produced cellulase, pectinase, xylanase, and nonspecific esterase when cultured in liquid medium. Moreover, R-5 multiplied in mineral medium containing cellulose, pectin, or xylan as a single carbon source. Thus, R-5 mycelia could degrade host cell walls at contact sites and probably utilize the degradation products as carbon sources. Received: May 16, 2002 / Accepted: July 9, 2002     

Improved growth of flathead flounder Hippoglossoides dubius in hypoxic waters in Funka Bay,Japan

To identify the factors that influence the growth rate of flathead flounder Hippoglossoides dubius in Funka Bay, Japan, we studied temporal changes in the growth of this species and its prey from 1989 to 2012. The lowest growth rate of H. dubius was recorded in the 1995 year-class, which had experienced severe hypoxia between 1995 and 1997. However, the highest growth rate was recorded in the 2007 year-class, which had experienced hypoxia between 2009 and 2011. In 2000 and 2001, small-sized flounder (<200 mm TL) from the 1995 year-class were feeding on mysidaceans, whereas those from other year-classes fed on small Ophiura spp. (<9 mm in disc diameter) in 2009, 2011, and 2012. Small Ophiura spp. were more abundant in 2007 and 2011 than in 2002 and 2003. Successful recruitment of Ophiura spp. took place between 2007 and 2011 and cohorts from these years could tolerate hypoxia. This suggests that food availability improved under hypoxic conditions, which led to improved growth in small flounders. Large flounders (≥200 mm TL) from all year-classes fed on prickleback fishes (Stichaeidae) and walleye pollock Gadus chalcogrammus juveniles. The growth of large flounders was enhanced by an increase in the abundance of Stichaeidae fishes and G. chalcogrammus juveniles.     

Serotonergic signals enhanced hamster sperm hyperactivation

In the present study, we investigated the regulatory mechanisms underlying sperm hyperactivation enhanced by 5-hydroxytryptamine (5-HT) in hamsters. First, we examined the types of 5-HT receptors that regulate hyperactivation. Hyperactivation was significantly enhanced by 5-HT_2A and 5-HT₄ receptor agonists. Moreover, the results of the motility assay revealed that 5-HT_2A, 5-HT₃, and 5-HT₄ receptor agonists significantly decreased the velocity and/or amplitude of sperm. Under 5-HT₂ receptor stimulation, hyperactivation was associated with phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP₃) receptor, soluble adenylate cyclase (sAC), and protein kinase A (PKA). In contrast, under 5-HT₄ receptor stimulation, hyperactivation was associated with transmembrane adenylate cyclase (tmAC), sAC, PKA, and CatSper channels. Accordingly, under the condition that sperm are hyperactivated, 5-HT likely stimulates PLC/IP₃ receptor signals via the 5-HT_2A receptor and tmAC/PKA/CatSper channel signals via the 5-HT₄ receptor. After sAC and PKA are activated by these stimulations, sperm hyperactivation is enhanced.     

The role of IFN-gamma in cattle infected with Theirelia sergenti

The spleen plays an important role in cellular immunity leading to parasite elimination, especially in hemo-protozoan parasite infection. In the present study, we quantified the serum IFN-gamma in splenectomized (SP) and non-splenectomized (NSP) cows infected with Theirelia sergenti (TS) to investigate whether the spleen plays a role in the Th1-type cytokine responses in cows following parasite infection. A transient increase in IFN-gamma was observed in the early stages of infection in the NSP cows, and the cows did not develop parasitemia. In contrast, the SP cows showed no IFN-gamma response at the early stage of infection, and the cows developed parasitemia following anemia. The NSP cows produced IgG1 and IgG2 antibodies to TS-specific, whereas the SP cows showed increases only in IgG1. Increased PBMC proliferation by cocanavalin A stimulation was observed concomitant with the IFN-gamma response. Pretreatment with IFN-gamma suppressed the propagation of TS in the SP cattle. These results suggest that the spleen plays an important role in the resistance to TS infection, leading to a Th1-type immune response. IFN-gamma might contribute to the activation of immunocytes and play an important role in the immune response to resist TS proliferation.     

Characterization of the neurotoxin produced by isolates associated with avian botulism

Several varieties of birds are affected by type C botulism. We conducted neutralization tests of culture supernatants of isolates from cases of avian botulism. Whereas the toxin produced by isolates derived from mammalian botulism was neutralized only with type C antitoxin, the toxins of all isolates related to avian botulism were neutralized with both type C and D antitoxins. An analysis of nucleotide sequences with several strains revealed that the neurotoxin gene in the isolates from avian botulism comprises two thirds of the type C neurotoxin gene and one third of the type D neurotoxin gene. This indicates that the neurotoxin of avian isolates is a mosaic of type C and D neurotoxins. We prepared three sets of primers to differentiate the gene for the mosaic form from the conserved genes of type C and D neurotoxins. The results of polymerase chain reaction with these primers indicated that all avian botulism-related isolates and specimens possess the gene for the mosaic form of the neurotoxin. The toxins purified from avian and mammalian isolates exhibited the same degree of lethality in mice, but the former showed greater toxicity to chickens than the latter. These results indicate that the mosaic neurotoxin is probably a pathogenic agent causing some forms of avian botulism.     

Variety in histochemical characteristics of the olfactory receptor cells in a flatfish, barfin flounder (Verasper moseri)

Variety in histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP9.5) and calretinin, and by lectin histochemistry with Phaseolus vulgaris leucoagglutinin (PHA-L) in the olfactory epithelium (OE) of the barfin flounder (Verasper moseri). PGP 9.5 immunoreactivity was observed in the ORC situated in the upper three fourths of the OE. Calretinin immunoreactivity was observed in the ORC which seemed to be immunonegative for PGP 9.5. These cells were located in the upper two thirds of the OE. PHA-L staining was observed in small subsets of the ORC. PGP 9.5 and calretinin immunoreactivities and PHA-L staining were also observed in the crypt cells unique to the fish OE. These findings suggest the different properties of olfactory perception among fish ORC.