Identification of heparin-binding proteins expressed on Trichosporon asahii cell surface.

Trichosporon asahii is a pathogenic yeast that cause trichosporonosis. T. asahii exhibits several colony morphologies, such as white (W)- or off-white (O)-type, which may affect virulence. In this study, we compared the expression pattern of heparin-binding proteins in various colony morphologies and identified heparin-binding protein in T. asahii. Surface plasmon resonance analysis revealed that cell surface molecules attached more strongly to heparin in W- than O-type cells. We purified and identified a heparin-binding protein strongly expressed in W-type cells using heparin-Sepharose beads, named it heparin-binding protein 1 (HepBP1), and expressed Flag-tagged HepBP1 in mammalian cells. The heparin-binding ability of Flag-tagged HepBP1 was confirmed by pulldown assay using heparin-Sepharose beads. Thus, HepBP1 is a heparin-binding protein on T. asahii cell surface. These results suggest that several T. asahii cell surface proteins interact with glycosaminoglycans; therefore, they could contribute to infection.

Hemagglutination ability and hemolytic activity of Trichosporon asahii.

Trichosporon asahii is a human fungal pathogen that causes deep-seated infections in immunocompromised patients. While the pathogenic mechanisms of T. asahii remain unknown, our previous studies indicate that adherent colony morphologies were generated from parent strains, which may contribute to their pathogenicity. In the present study, we analyzed the hemolytic and hemagglutination activities of T. asahii. We report that T. asahii cells demonstrate hemagglutination and hemolytic activities, and that cell surface molecules play a role in the hemagglutination activity of adherent strains. These observations suggest that hemagglutination and hemolysis may be one of the pathogenic mechanisms of T. asahii.

Pyridoxamine: A novel treatment for schizophrenia with enhanced carbonyl stress.

AIM: The aim of this clinical trial was to obtain proof of concept for high-dose pyridoxamine as a novel treatment for schizophrenia with enhanced carbonyl stress. METHODS: Ten Japanese schizophrenia patients with high plasma pentosidine, which is a representative biomarker of enhanced carbonyl stress, were recruited in a 24-week, open trial in which high-dose pyridoxamine (ranging from 1200 to 2400 mg/day) was administered using a conventional antipsychotic regimen. Main outcomes were the total change in Positive and Negative Syndrome Scale score and the Brief Psychiatric Rating Scale score from baseline to end of treatment at week 24 (or at withdrawal). RESULTS: Decreased plasma pentosidine levels were observed in eight patients. Two patients showed marked improvement in their psychological symptoms. A patient who harbors a frameshift mutation in the Glyoxalase 1 gene also showed considerable reduction in psychosis accompanied with a moderate decrease in plasma pentosidine levels. A reduction of greater than 20% in the assessment scale of drug-induced Parkinsonism occurred in four patients. Although there was no severe suicide-related ideation or behavior, Wernicke's encephalopathy-like adverse drug reactions occurred in two patients and were completely suppressed by thiamine supplementation. CONCLUSION: High-dose pyridoxamine add-on treatment was, in part, effective for a subpopulation of schizophrenia patients with enhanced carbonyl stress. Further randomized, placebo-controlled trials with careful monitoring will be required to validate the efficacy of high-dose pyridoxamine for these patients.

Cell surface hydrophobicity and colony morphology of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic basidiomycetous yeast. Individual strains of T. asahii have different colony morphologies. However, it is not clear whether cell surface phenotypes differ among the colony morphologies. Here we characterized the cell surface hydrophobicity and analysed the carbohydrate contents of the cell surface polysaccharides in T. asahii clinical isolates with various colony morphologies. Among the three distinctive colony morphologies obtained from one clinical isolate, the white-type morphology exhibited higher hydrophobicity. The hydrophobicity of heat-killed T. asahii cells was greatly reduced after periodate oxidation of the cell surface carbohydrates. Furthermore, the cell wall and extracellular polysaccharide components differed among the morphologies. Our results suggest that T. asahii cell surface hydrophobicity is affected by cell surface carbohydrate composition. Copyright © 2016 John Wiley & Sons, Ltd.

Growth Inhibition of an Opportunistic Yeast Pathogen Trichosporon asahii by Staphylococcus epidermidis.

In the co-culture of Staphylococcus epidermidis and Trichosporon asahii, a fungal pathogen, it was observed that live S. epidermidis inhibited the growth of T. asahii. Soluble active anti-T. asahii substances were speculated to be produced by S. epidermidis in culture medium. Using 1H- and 13C-NMR spectra and electron ionization-high resolution mass spectrometry (HR-negative-FAB-MS), we separated the active molecule and identified it as lactic acid. Commercially available L-lactic acid and D-lactic acid inhibited the growth of T. asahii. These results show that metabolites from bacterial populations are involved in the interactions of pathogenic fungi. The use of antibacterial agents to treat primary diseases could lead to the disruption of normal microbial communities and could cause opportunistic infections such as trichosporonosis.

The regulation of soluble receptor for AGEs contributes to carbonyl stress in schizophrenia.

Our previous study showed that enhanced carbonyl stress is closely related to schizophrenia. The endogenous secretory receptor for advanced glycation end-products (esRAGE) is a splice variant of the AGER gene and is one of the soluble forms of RAGE. esRAGE is considered to be a key molecule for alleviating the burden of carbonyl stress by entrapping advanced glycation end-products (AGEs). In the current study, we conducted genetic association analyses focusing on AGER, in which we compared 212 schizophrenic patients to 214 control subjects. We also compared esRAGE levels among a subgroup of 104 patients and 89 controls and further carried out measurements of total circulating soluble RAGE (sRAGE) in 25 patients and 49 healthy subjects. Although the genetic association study yielded inconclusive results, multiple regression analysis indicated that a specific haplotype composed of rs17846798, rs2071288, and a 63 bp deletion, which were in perfect linkage disequilibrium (r2 = 1), and rs2070600 (Gly82Ser) were significantly associated with a marked decrease in serum esRAGE levels. Furthermore, compared to healthy subjects, schizophrenia showed significantly lower esRAGE (p = 0.007) and sRAGE (p = 0.03) levels, respectively. This is the first study to show that serum esRAGE levels are regulated by a newly identified specific haplotype in AGER and that a subpopulation of schizophrenic patients are more vulnerable to carbonyl stress.

Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population.

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.

Switching of colony morphology and adhesion activity of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic yeast that causes trichosporonosis, a deep-seated infection, in immunocompromised hosts. Pathogenic factors involved in this infection have not been investigated in detail, but morphological phenotype switching is thought to be important for T. asahii pathogenesis. Therefore, we analyzed adhesion, which may be a key early step in T. asahii infection, after morphological phenotype switching. T. asahii clinical isolates show several colony morphologies. In this study, colonies showing white-farinose (W), off-white-smooth (O), off-white-rugose (OR), smooth (S), and yellowish-white (Y) morphologies were obtained from three isolates and compared in an adhesion assay performed in cell culture dishes. At least one type of colony morphology from each clinical isolate adhered strongly to the culture dish surface, although the colony type that displayed strong adherence varied among the strains. Thus, morphological phenotype switching altered the adhesion of T. asahii strains.

Interaction of surface molecules on Cryptococcus neoformans with plasminogen.

Microbial pathogens are known to express molecules that interact with host proteins, leading to invasion and colonization. For example, some pathogenic microorganisms express proteins that bind to and enhance the activity of plasminogen. In this way, pathogens utilize the host fibrinolytic system to promote invasion. We found that triosephosphate isomerase (TPI), a glycolytic enzyme produced by Staphylococcus aureus, bound to mannooligosaccharides from the pathogenic capsulated fungus Cryptococcus neoformans and human plasminogen, suggesting that TPI is a moonlighting protein. Several C. neoformans surface proteins are thought to be plasminogen-binding proteins. Here, we examined the ability of surface polymers (including polysaccharides) to bind plasminogen. Heat-killed C. neoformans cells transformed plasminogen into plasmin in a dose-dependent manner in the presence of tissue plasminogen activator. Soluble polysaccharides were found to bind plasminogen based on surface plasmon resonance (SPR) analysis. Neutral polysaccharides fractionated using DEAE column chromatography bound and activated plasminogen. However, the fraction containing glucuronoxylomannan (the primary component of the capsule) did not activate plasminogen. In addition, binding between glucuronoxylomannan and plasminogen was weak. Components of the neutral polysaccharides were identified as mannose, galactose, glucose and xylose. In conclusion, neutral polysaccharides that may affect fibrinolysis were detected on the surface of C. neoformans.

Detection and characterization of plasminogen receptors on clinical isolates of Trichosporon asahii.

Trichosporon asahii is the major causative agent of deep-seated trichosporonosis. The virulence factors of this yeast pathogen remain uncharacterized. To investigate the pathogenicity of T. asahii, we focused on the interactions between surface molecules of the yeast and host biomolecules. We examined the ability of surface molecules to bind human plasminogen using clinical isolates of T. asahii. Living T. asahii cells accelerated the conversion of plasminogen to plasmin in a dose-dependent manner in the presence of tissue plasminogen activator. Extracts from cells using lithium chloride contained plasminogen-binding molecules based on surface plasmon resonance (SPR) analyses. In all strains tested, several of the fractions obtained using DEAE column chromatography bound and accelerated the conversion of plasminogen to plasmin. Based on far-Western blotting analyses, a common protein was identified within the four strains, which was identified as a hypothetical protein from genome analyses of T. asahii. blast searches suggested the protein might be heparinase, and heparinase activity was detected in the T. asahii extract. Furthermore, affinity chromatography using plasminogen as a ligand detected one protein band by SDS-PAGE, which was identified as thioredoxin-dependent peroxide reductase.SPR analyses suggested the presence of molecules on T. asahii cells that could bind plasminogen with differing affinities.

Interaction of Host Proteins with Cell Surface Molecules of the Pathogenic Yeast Trichosporon asahii.

Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the factors that contribute to the occurrence of T. asahii infections remain unclear. Since adhesins are typical virulence factors reported for pathogenic fungi, we looked for host proteins that interact with the T. asahii cell surface. T. asahii and Candida albicans were used for screening using a pull-down assay with fetal bovine serum. Serum albumin and elongation factor 2 were identified as the yeast-binding serum proteins. Additionally, we investigated the interactions of the cell surface-associated molecules (CSM) of T. asahii with vitronectin (VTN), fibronectin, fetuin-A, and alpha-1antitrypsin (AAT). The surface plasmon resonance (SPR) method was used to examine the interaction between CSM and human proteins. On the other hand, the pull-down assay was used to examine the interaction between human proteins and the T. asahii cell surface. Serum albumin, AAT, and VTN were found to interact with T. asahii in both SPR and pull-down assays. This study identified several proteins that interact with T. asahii, suggesting that these proteins play a role in infection mechanisms.

Schizophrenia: maternal inheritance and heteroplasmy of mtDNA mutations.

Role of mitochondrial pathology in schizophrenia has not been fully clarified. We searched for distinctive variants in mtDNA extracted from the gray matter of postmortem brains and from peripheral blood samples. We screened mtDNA region containing 5 genes encoding subunits of cytochrome c oxidase and ATPases. Polymorphisms not already reported in databases are recorded as unregistered rare variants. Four unregistered, non-synonymous rare variants were detected in 4 schizophrenic samples. Seven registered non-synonymous variants were not previously detected in non-psychotic Japanese samples registered in the mtSNP database. These variants may contribute to disease pathophysiology. In one family, compound mutations showed co-segregation with schizophrenia. MtDNA mutations could confer a risk for schizophrenia in the Japanese population, although further analyses are needed.

Paradox of schizophrenia genetics: is a paradigm shift occurring?

BACKGROUND: Genetic research of schizophrenia (SCZ) based on the nuclear genome model (NGM) has been one of the most active areas in psychiatry for the past two decades. Although this effort is ongoing, the current situation of the molecular genetics of SCZ seems disappointing or rather perplexing. Furthermore, a prominent discrepancy between persistence of the disease at a relatively high prevalence and a low reproductive fitness of patients creates a paradox. Heterozygote advantage works to sustain the frequency of a putative susceptibility gene in the mitochondrial genome model (MGM) but not in the NGM. METHODS: We deduced a criterion that every nuclear susceptibility gene for SCZ should fulfill for the persistence of the disease under general assumptions of the multifactorial threshold model. SCZ-associated variants listed in the top 45 in the SZGene Database (the version of the 23rd December, 2011) were selected, and the distribution of the genes that could meet or do not meet the criterion was surveyed. RESULTS: 19 SCZ-associated variants that do not meet the criterion are located outside the regions where the SCZ-associated variants that could meet the criterion are located. Since a SCZ-associated variant that does not meet the criterion cannot be a susceptibility gene, but instead must be a protective gene, it should be linked to a susceptibility gene in the NGM, which is contrary to these results. On the other hand, every protective gene on any chromosome can be associated with SCZ in the MGM. Based on the MGM we propose a new hypothesis that assumes brain-specific antioxidant defenses in which trans-synaptic activations of dopamine- and N-methyl-d-aspartate-receptors are involved. Most of the ten predictions of this hypothesis seem to accord with the major epidemiological facts and the results of association studies to date. CONCLUSION: The central paradox of SCZ genetics and the results of association studies to date argue against the NGM, and in its place the MGM is emerging as a viable option to account for genomic and pathophysiological research findings involving SCZ.

[Schizophrenia and carbonyl stress].

Glyoxalase I (GLO1) is an enzyme essential for the cellular detoxification of reactive carbonyl compounds. Its impairment leads to carbonyl stress and the eventual formation of advanced glycation end products (AGEs) including pentosidine. In the present study, we provide for the first time evidence of genetic abnormalities in GLO1 with their attendant derangements of biochemistry in a small population of schizophrenia. We found that seventeen schizophrenia (approximately 17.4%) showed increases in plasma pentosidine content although they have no physical complications, and concomitantly marked decrease of pyridoxal levels was found in schizophrenia. Vitamin B6 exists in three forms, i.e., pyridoxine, pyridoxal and pyridoxamine. Pyridoxamine has potent ability to entrap RCOs and prevent their toxicity. Further study of profiling of metabolomics and clinical manifestations in schizophrenia could pave the way for novel, better therapeutic/preventive measures for this devastating disease.

[Carbonyl stress-related schizophrenia--perspective on future therapy and hypotheses regarding pathophysiology of schizophrenia].

Various factors are thought to be involved in the pathogenesis of schizophrenia. Recently, biochemical studies using human samples and animal models suggest that oxidative stress and the resulting formation of reactive carbonyl compounds (RCOs) contribute to the pathophysiology of schizophrenia. The accumulation of RCOs, carbonyl stress, results in the modification of proteins and formation of advanced glycation end products (AGEs), such as pentosidine. We previously reported that a certain subtype of schizophrenic patients exhibit idiopathic carbonyl stress with high plasma pentosidine levels and the depletion of vitamin B6, without underlying diabetes or chronic kidney disease, the two major causes of elevated AGEs. Agents able to inhibit AGE formation or entrap carbonyl compounds may also prove to be of therapeutic value. Pyridoxamine, a non-toxic, water-soluble vitamin B6, has potent abilities to entrap toxic carbonyl compounds and prevent toxicity. In particular, the markedly lowered vitamin B6 levels in schizophrenic patients with high pentosidine levels suggest that pyridoxamine may prove to be clinically useful.

[Molecular pathophysiology of schizophrenia and preventive strategy in pubertal period].

A novel frameshift mutation in glyoxalase 1 (GLO1) gene was detected in a patient with schizophrenia of a pedigree with multiple affected individuals. The patient carrying the mutation showed decreased enzymatic activity by 50%, 3.7 times high level of advanced glycation end products (AGEs) that is substrate of GLO1 and 20% of serum vitamin B6 compared to controls. Case-control study of GLO1 gene suggested that Ala allele of Glu111Ala was associated with schizophrenia. In vitro study using COS-7 cells transfected with cDNA of GLO1 yielded that enzymatic activity is lower in GLO1 with Ala111 than that of Glu111. The homozygotes of Ala111 showed 16% decreased GLO1 activities in RBC as compared with that of Glu111/Ala111 and Glu111/Glu111. Plasma AGEs levels were significantly high and serum vitamin B6 was significantly low in 45 schizophrenics than that of 61 control subjects. Supplementation of vitamin B6 to cases with the genetic defect of GLO1 before onset of psychosis is suggested to be possible strategy for prevention of schizophrenia until pubertal stage since such mutation carriers could have been exposed by high level of AGEs for a long time before disease onset.

Cooperation of LIM domain-binding 2 (LDB2) with EGR in the pathogenesis of schizophrenia.

Genomic defects with large effect size can help elucidate unknown pathologic architecture of mental disorders. We previously reported on a patient with schizophrenia and a balanced translocation between chromosomes 4 and 13 and found that the breakpoint within chromosome 4 is located near the LDB2 gene. We show here that Ldb2 knockout (KO) mice displayed multiple deficits relevant to mental disorders. In particular, Ldb2 KO mice exhibited deficits in the fear-conditioning paradigm. Analysis of the amygdala suggested that dysregulation of synaptic activities controlled by the immediate early gene Arc is involved in the phenotypes. We show that LDB2 forms protein complexes with known transcription factors. Consistently, ChIP-seq analyses indicated that LDB2 binds to > 10,000 genomic sites in human neurospheres. We found that many of those sites, including the promoter region of ARC, are occupied by EGR transcription factors. Our previous study showed an association of the EGR family genes with schizophrenia. Collectively, the findings suggest that dysregulation in the gene expression controlled by the LDB2-EGR axis underlies a pathogenesis of subset of mental disorders.

LDB2 locus disruption on 4p16.1 as a risk factor for schizophrenia and bipolar disorder.

We had previously reported the case of a male patient with schizophrenia, having de-novo balanced translocation. Here, we determined the exact breakpoints in chromosomes 4 and 13. The breakpoint within chromosome 4 was mapped to a region 32.6 kbp upstream of the LDB2 gene encoding Lim domain binding 2. Variant screening in LDB2 revealed a rare novel missense variant in patients with psychiatric disorder.

Adherence and Cytotoxicity of Candida spp. to HaCaT and A549 cells.

Candida species are opportunistic fungal pathogens that cause superficial or invasive infections. Recently, the incidence of infection by non-Candida albicans species, especially Candida glabrata, has increased. In this study, we analyzed the adhesion and cytotoxicity of various Candida spp. that are part of the normal human microbiota. C. albicans adheres well to cell culture plates and to cultured cells. C. glabrata selectively adheres to epithelial cells rather than to cell culture plates. Candida parapsilosis insufficiently adheres to confluent monolayers of human lung epithelial A549 and keratinocyte HaCaT cells. We then analyzed the cytotoxicity of C. albicans and C. glabrata, which adhered well to epithelial cells. C. glabrata has been found to cause more damage to A549 cells than to HaCaT cells, suggesting that resident Candida spp. have distinct cytotoxic effects in different tissues. It is important to clarify the properties of Candida spp. as there is evidence that normal microbiota can cause infections. Our data suggest that it is necessary to use appropriate cell lines for characterizing the adherence and cytotoxicity of Candida spp.

DeltaNp63/BMP-7-dependent expression of matrilin-2 is involved in keratinocyte migration in response to wounding.

p63 is expressed as multiple variants including TA and DeltaN forms. Since p63-deficient mice displayed profound defects of stratified epithelia, p63 is an essential transcription factor required for epidermal morphogenesis. However, precise molecular mechanisms behind contribution of p63 to normal skin formation and healing skin wounds remained unclear. In this study, we demonstrated that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of extracellular matrix protein matrilin-2. Knocking down of DeltaNp63 in human keratinocyte HaCaT cells led to a significant reduction of matrilin-2. Intriguingly, BMP-7 which is one of DeltaNp63-target gene products, induced matrilin-2 and attenuated inhibitory effect of siRNA against DeltaNp63 on matrilin-2. Furthermore, a remarkable cell migration in response to wounding took place in DeltaNp63- or matrilin-2-knocked down cells. Taken together, our present findings indicate that DeltaNp63/BMP-7 signaling pathway modulates wound healing process through the regulation of matrilin-2.