Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae) reveals a new genus Turmalinea and highlights the utility of ITS minisatellite-like insertions for molecular identification.

The sequestrate (truffle-like) basidiomycete genera Rossbeevera, Chamonixia, and Octaviania are closely related to the epigeous mushroom genera Leccinum and Leccinellum. In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of Rossbeevera and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, EF-1α) and two mitochondrial DNA loci (ATP6 and mtSSU) as well as precise morphological observations. Phylogenetic analyses of three nuclear loci suggest a complex evolutionary history with sequestrate fruiting bodies present in several clades, including a previously unrecognized sister clade to Rossbeevera. Here we propose a new sequestrate genus, Turmalinea, with four new species and one new subspecies as well as two new species of Rossbeevera. The three-locus nuclear phylogeny resolves species-level divergence within the Rossbeevera-Turmalinea lineage, whereas a separate phylogeny based on two mitochondrial genes corresponds to geographic distance within each species-level lineage and suggests incomplete lineage sorting (ILS) and gene introgression within several intraspecific lineages of Rossbeevera. Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within Rossbeevera probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all Rossbeevera and Turmalinea species. A barcode gap analysis demonstrates that the insertion is more informative for discrimination at various taxonomic levels than the rest of the ITS region and could therefore serve as a unique molecular barcode for these genera.

Taxonomic and life cycle reappraisals of the marine basidiomycete Nia vibrissa complex, with descriptions of three new Nia species.

The marine basidiomycete Nia vibrissa has been regarded as a species complex, possibly including several species, because morphological variations in fruitbody, spore, and spore appendage have been observed in materials from worldwide collections. Using more than 50 monosporic isolates of N. vibrissa-like fungi mainly obtained from Japanese beach coasts, we investigated their molecular phylogeny, morphological characteristics, mating compatibility, nuclear behavior during spore formation, and life cycles. Molecular phylogenetic analyses separated the examined strains into seven clades. Each clade of fungi exhibited distinctive characteristics in fruitbodies and spores produced by culturing monokaryotic strains and mated dikaryotic strains; these characteristics included the color of fruitbodies, apical structure of peridial hair hyphae, spore shape, and apical structure of spore appendages. Mating tests of monokaryotic strains demonstrated mating compatibility between strains within a clade and incompatibility among clades. Therefore, each clade of fungi was phylogenetically, morphologically, and biologically recognized as a different Nia species. Observation of the type specimen of N. vibrissa revealed a tiny T-shaped apical structure of spore appendages-not mentioned in the original description-that is unique to the species. This finding, together with the original description, suggests that our studied strains include N. aff. vibrissa, whose morphology is mostly identical to N. vibrissa sensu stricto, and three new species. Thus, we describe three new Nia species and propose emendation of the descriptions of the genus Nia. Culture-based studies have demonstrated that Nia species have both sexual and asexual morphs that produce morphologically similar fruitbodies (basidiomata and conidiomata) and spores (basidiospores and conidia). Because it has both morphs forming appendaged waterborne basidiospores and conidia, Nia must be the most well-adapted marine basidiomycete, ensuring the continuation of new generations by two morphs, while distributing in and inhabiting numerous marine environments.

Prostaglandin-related immune suppression in cattle.

Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.

The expression of osteopontin is increased in vessels with blood-brain barrier impairment.

AIMS: We previously reported that the blood-brain barrier (BBB) function was deteriorated in vessels located along hippocampal fissures in stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we examined changes of gene expression in the BBB-damaged vessels of SHRSP. METHODS: Vascular samples were microdissected from the hippocampi of SHRSP and Wistar-Kyoto (WKY) as a control and the difference in gene expression between the BBB-damaged vessels in SHRSP and vessels without BBB damage in WKY was examined by a microarray. The differences in gene and protein expression between brain tissues in the two strains of rats were examined using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. RESULTS: The microarray assay revealed that the ratio of osteopontin gene expression in the vascular tissue of the hippocampi of SHRSP to that of WKY was the highest among 8435 genes. Real-time RT-PCR analysis revealed that the gene expression of osteopontin was significantly increased in the hippocampal samples of SHRSP compared with that in the hippocampal samples of WKY rats or with that in the cortical samples of SHRSP. Immunohistochemical and Western blot analyses showed that the osteopontin protein expression was seen in perivascular ED1-positive macrophages/microglial cells located around hippocampal fissures and significantly increased in the hippocampi of SHRSP compared with that of WKY. CONCLUSIONS: These findings indicate that the expression of osteopontin is increased in BBB-damaged vessels in hypertensive SHRSP compared with that in vessels without BBB impairment in WKY rats, suggesting a role for osteopontin in BBB function.

Tumor necrosis factor-alpha (TNF-alpha) plays a protective role in acute viralmyocarditis in mice: A study using mice lacking TNF-alpha.

BACKGROUND: It has been reported that tumor necrosis factor-alpha (TNF-alpha) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-alpha on viral myocarditis, however, have not been fully elucidated. METHODS AND RESULTS: To investigate the role of TNF-alpha in the progression of viral myocarditis, we used TNF-alpha gene-deficient mice (TNF-alpha(-/-)) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-alpha(-/-) mice after EMCV infection was significantly lower than that of TNF-alpha(+/+) mice (0% versus 67% on day 14). Injection of recombinant human TNF-alpha (0.2 to 4.0 microg/mouse IV) improved the survival of TNF-alpha(-/-) mice in a dose-dependent manner, indicating that TNF-alpha is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-alpha(-/-) mice compared with TNF-alpha(+/+) mice. CONCLUSIONS: These observations suggested that TNF-alpha is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-alpha plays a protective role in the acute stage of viral myocarditis.

Enhancement of Fc epsilon RII/CD23 expression on U937 cells with opsonized zymosan: the requirement of a Fc gamma RI/CD64 mediated signal associated phagocytosis.

The kinetics of surface Fc epsilon RII/CD23 was tested on monocytic cell line U937 stimulated with opsonized zymosan. Zymosan opsonized with human serum enhanced not only the expression of surface Fc epsilon RII/CD23 but also Fc epsilon RII/CD23 mRNA detected by Northern blot and in situ hybridization techniques. This stimulation showed a marked synergism with IL-4 in the induction of Fc epsilon RII/CD23. Heat-inactivation of serum did not affect the inducibility of Fc epsilon RII/CD23 by opsonized zymosan, suggesting the involvement of serum substances other than complement. Zymosan treated with human gamma-globulin also induced Fc epsilon RII/CD23, indicating the possible involvement of Fc gamma receptors. The Fc epsilon RII/CD23 inducing effect of opsonized zymosan was partially blocked by pretreatment with heat-aggregated human gamma-globulin or an anti-Fc gamma RI monoclonal antibody but not by the anti-Fc gamma RII or Fc gamma RIII antibody. Our results showed the involvement of signals from Fc gamma receptor associated phagocytosis in the induction of Fc epsilon RII/CD23.

Evaluation of left atrial appendage stasis in patients with atrial fibrillation using transesophageal echocardiography with an intravenous albumin-contrast agent.

To assess stasis in the left atrial appendage in patients with atrial fibrillation and to identify patients at increased risk for thromboembolism, we performed transesophageal echocardiography with an intravenous albumin contrast agent (Albunex) in 25 patients with atrial fibrillation and in 22 patients in sinus rhythm. We demonstrated that the absence of opacification in the left atrial appendage after Albunex administration implies a high risk of left atrial thrombus and cardiogenic thromboembolism.

Induction and function of Fc epsilon RII on YT cells; possible role of ADF/thioredoxin in Fc epsilon RII expression.

The regulation of low-affinity Fc receptor for IgE (Fc epsilon RII) and the characteristics of both membrane and soluble forms of Fc epsilon RII were studied using YT cell line. We found that YT cells, a human NK like cell line, expressed Fc epsilon RII after IL-1 stimulation. Cross-linking of Fc epsilon RII on IL-1-stimulated YT cells as well as the transfectant of Fc epsilon RII-cDNA (YTSER) resulted in the up-regulation of IL-2R alpha (p55/Tac). A 59 kDa protein phosphorylated at tyrosine residues was co-immunoprecipitated with Fc epsilon RII from YTSER lysate using H107 anti-Fc epsilon RII mAb. YTSER not only expressed Fc epsilon RII on their surface but also secreted soluble form of Fc epsilon RII (sFc epsilon RII/sCD23; IgE binding factor). Affinity purification revealed that sFc epsilon RII released from YTSER is heterogeneous and consisted of several proteins differing in molecular weight. Both EBV+ B cells and HTLV-1+ T cells are high producers of ATL derived factor (ADF)/thioredoxin (TRX) and express Fc epsilon RII and IL-2R alpha respectively. To clarify the mechanism of Fc epsilon RII and IL-2R alpha induction by ADF/TRX, we examined the effect of ADF/TRX on the bindability of nuclear factor kappa B (NF-kappa B), which is known to regulate IL-2R alpha gene expression. In the gel shift assay, ADF/TRX was shown to enhance the bindability of NF-kappa B to its responsive element.

Application of electrospray ionization MS/MS and matrix-assisted laser desorption/ionization-time of flight mass spectrometry to structural analysis of the glycosyl-phosphatidylinositol-anchored protein.

We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.

A cooperative study of sarcoidosis in Asia and Africa: descriptive epidemiology.

An eight-nation cooperative epidemiological study revealed the Asian and African features of sarcoidosis. Almost every country reported from several to less than 30 cases, except for Japan which had already collected over 3,000 cases. Not a single case was found in the mass x-ray surveys conducted by several countries on a large scale (Tables 1 and 2). Although the number of the cases included in this study were small, this information is the first of this kind for Asia and Africa.

Improvement of gas exchange following endobronchial instillation of an exogenous surfactant in an infant with respiratory failure by postoperative pulmonary haemorrhage.

We administered surfactant to a 5-month-old infant with respiratory failure due to right pulmonary haemorrhage accompanied by oedema following abdominal surgery. These pathological conditions were probably precipitated by disseminated intravascular coagulation and intra-operative excessive administration of fluids, respectively. Endobronchial instillation of the exogenous surfactant (120 mg) after selective intubation of the right bronchus produced a dramatic improvement of gas exchange 30 min after treatment and of chest X-ray findings at 6 h post-treatment. This case on an infant indicates that administration of surfactant may be one of promising therapeutic approaches to respiratory failure due to pulmonary haemorrhage.

Inhibitory effect of prostaglandin E1 on human neutrophil function.

Neutrophils accumulated in the lung are thought to play a pivotal role in the pathogenesis of host auto-injury such as adult respiratory distress syndrome (ARDS). We investigated the effect of prostaglandin E1 (PGE1) on several aspects of human neutrophil function. PGE1 significantly decreased reactive oxygen species (ROS), (O2-, H2O2, OH.) generation by neutrophils as well as neutrophil phagocytosis and chemotaxis. In contrast, the drug did not affect the levels of ROS generated by a cell-free ROS generating system. In addition, intracellular calcium concentrations ([Ca2+]i) in neutrophils stimulated by f-Met-Leu-Phe were decreased in the presence of PGE1. These data suggest that the reduction in ROS production and neutrophil phagocytosis and chemotaxis by PGE1 may contribute to the effectiveness of the drug in host auto-injury including ARDS. The suppression of the increase in [Ca2+]i may at least be responsible for inhibition of these neutrophil functions by PGE1.

Secondary diabetic retinopathy in chronic pancreatitis.

We found abnormal glucose tolerance curves in 45 of 47 patients with chronic pancreatitis and observed secondary diabetic retinopathy in eight of 45 cases showing slight changes in the fundus. Abnormal glucose tolerance curves were somewhat related to the exocrine dysfunction of the pancreas. Slightly abnormal glucose tolerance curves were observed frequently in patients with chronic pancreatitis, and both insulin and glucagon responses were decreased. We could not explain the cause of the low frequency of secondary diabetic retinopathy in pancreatic diabetes from the results of insulin and glucagon response tests.

Activation of Fc epsilon RI-positive eosinophils in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune disease frequently occurring in elderly persons. It has been reported that 92-kDa gelatinase released from eosinophils cleaves the extracellular domain of BP180 protein, suggesting a direct role of eosinophils in bulla formation in this disease. The expression of the high-affinity IgE receptor, Fc epsilon RI, on eosinophils was examined in patient with BP. Samples of affected skin obtained from 7 patients with BP were stained immunohistochemically by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method and mirror sections were examined. Eosinophils were present at a rate of 1.0-19.0% in lesions of the dermis, and the number of IgE-positive cells exceeded that of Fc epsilon RI-positive cells in all cases. These cells were not detected in the epidermis, and examination of mirror sections confirmed that the Fc epsilon RI-positive cells corresponded to eosinophils. It has been demonstrated that Fc epsilon RI-positive cells are involved in the dermal lesions of BP. The activation of eosinophils by Fc epsilon RI may participate in the pathogenesis of BP by triggering the degranulation of mast cells.

Detection of lysyl oxidase gene expression in rat skin during wound healing.

Lysyl oxidase (LOX) initiates the crosslinking of the lysine-derived aldehyde and plays an essential role in maturation of collagen, for example in wound healing. Although the activity of this enzyme has been examined in various disorders, and a further intriguing aspect of the relationship between LOX and tumorigenesis has recently emerged, its gene expression pattern in tissues is still unknown. We examined LOX gene expression during wound healing in rat skin. In addition, type III collagen gene expression was studied to determine the formation of fibrils. The LOX mRNA level reached a peak by day 3 after injury, which was earlier than that of type III collagen, and continued at a high level until day 22. The type III collagen mRNA level began to rise from day 3 and had increased intensely by day 22. In situ hybridization revealed grains corresponding to LOX mRNA in the fibroblasts of the granulomatous tissue. These results suggest that LOX is produced before collagen synthesis in preparation for crosslinking in the early phase of wound healing.

3-Hydroxyanthranilic acid, an L-tryptophan metabolite, induces apoptosis in monocyte-derived cells stimulated by interferon-gamma.

3-Hydroxyanthranilic acid (3-HAA), a metabolite of L-tryptophan, accumulates in monocyte-derived cells (THP-1), but not in other cell lines tested (MRC-9, H4, U373MG, Wil-NS), following immune stimulation that induces indoleamine-2,3-dioxygenase (IDO), a rate-limiting enzyme in the L-tryptophan kynurenine pathway. We examined whether metabolites of the L-tryptophan-kynurenine pathway act to induce apoptosis in monocytes/macrophages. Of the L-tryptophan metabolites tested, only 3-HAA at a concentration of 200 micromol/L was found to induce apoptosis in THP-1 and U937 cells. The addition of ferrous or manganese ions further enhanced apoptosis and free radical formation by 3-HAA in these two types of cells. The apoptotic response induced by 3-HAA was significantly attenuated by the addition of antioxidant, alpha-tocopherol or Trolox (a water-soluble analogue of vitamin E), and the xanthine oxidase inhibitor, allopurinol. In addition, the 3-HAA-induced apoptotic response was slightly attenuated by catalase, but not by superoxide dismutase (SOD), indicating that generation of hydrogen peroxide is involved in this response. Interferon-gamma (IFN-gamma), an inducer of IDO, potently induced apoptosis in THP-1 cells, but not in U937 cells, in the presence of ferrous or manganese ions. This different susceptibility to apoptosis inducer between THP-1 and U937 cells may depend on the capacity of the cells for 3-HAA synthesis following IDO induction by IFN-gamma. Furthermore, apoptosis was suppressed by cycloheximide in THP-1 cells, suggesting that newly synthesized proteins may be essential for apoptotic events. These results suggest that 3-HAA induces apoptosis in monocytes/macrophages under inflammatory or other pathophysiological conditions.

Pedoscope studies on neonatal activity and center of gravity after delivery.

Seventeen normal term infants delivered at the Jikei University School of Medicine were placed daily on a pedoscope in the supine and prone position after birth, and the movement of the gravity center and changes in the activities of the extremities were assessed. The results indicated that both the activity of the extremities and the movement of the gravity center were sluggish after birth, that both increased to reach peaks within 1 to 3 days, and that then temporary decreases occurred before they increased again. The probable reason for the temporary decrease in activity may be habituation or a decrement in the infant, and it is also presumed that the increase in activity after the transitory decrease reflects natural development.

Significant reduction of 125 I-meta-iodobenzylguanidine accumulation directly caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine, a toxic agent for inducing experimental Parkinson's disease.

A significant reduction of cardiac 123I-meta-iodobenzylguanidine (MIBG) accumulation has been reported in patients with idiopathic Parkinson's disease. However, it is unclear whether this reduction in cardiac sympathetic nerve is caused primarily or secondarily to the degeneration of sympathetic nerve centres which occurs in Parkinson's disease. Therefore, we examined neuronal 125I-MIBG accumulation in mice hearts of an experimental Parkinson's disease model and in sympathetic cells without any neuronal innervation. Cardiac accumulation of 125I-MIBG was determined 4h after intravenous injection of 125I-MIBG in mice pretreated with 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP), an inducer of Parkinson's disease. In an in vitro study, uptake of 125I-MIBG was determined in a cultured pheochromocytoma cell line (PC-12), which was pretreated with MPTP. MPTP reduced MIBG accumulation mainly in its neuronal component of mice hearts, suggesting that MPTP impairs cardiac sympathetic nerves to uptake MIBG. Application of MPTP also caused near-complete blockade of 125I-MIBG accumulation in PC-12 cells. In the experimental PD models, it was shown that neuronal accumulation of MIBG was impaired by the direct action of MPTP to the sympathetic cells. These findings support the idea that cardiac sympathetic nerves are primarily impaired in Parkinson's disease despite the presence or absence of systemic autonomic failure.