Arabidopsis MAPKKK δ-1 is required for full immunity against bacterial and fungal infection.

The genome of Arabidopsis encodes more than 60 mitogen-activated protein kinase kinase (MAPKK) kinases (MAPKKKs); however, the functions of most MAPKKKs and their downstream MAPKKs are largely unknown. Here, MAPKKK δ-1 (MKD1), a novel Raf-like MAPKKK, was isolated from Arabidopsis as a subunit of a complex including the transcription factor AtNFXL1, which is involved in the trichothecene phytotoxin response and in disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A MKD1-dependent cascade positively regulates disease resistance against PstDC3000 and the trichothecene mycotoxin-producing fungal pathogen Fusarium sporotrichioides. MKD1 expression was induced by trichothecenes derived from Fusarium species. MKD1 directly interacted with MKK1 and MKK5 in vivo, and phosphorylated MKK1 and MKK5 in vitro. Correspondingly, mkk1 mutants and MKK5RNAi transgenic plants showed enhanced susceptibility to F. sporotrichioides. MKD1 was required for full activation of two MAPKs (MPK3 and MPK6) by the T-2 toxin and flg22. Finally, quantitative phosphoproteomics suggested that an MKD1-dependent cascade controlled phosphorylation of a disease resistance protein, SUMO, and a mycotoxin-detoxifying enzyme. Our findings suggest that the MKD1-MKK1/MKK5-MPK3/MPK6-dependent signaling cascade is involved in the full immune responses against both bacterial and fungal infection.

C-terminal residues of ferredoxin-NAD(P)+ reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP+/NADPH.

Ferredoxin-NAD(P)+ reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP+ and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP+ and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326-Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337-Glu360 and Ser338-Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP+/NADPH.

Identification of sperm equatorial segment protein 1 in the acrosome as the primary binding target of peanut agglutinin (PNA) in the mouse testis.

Peanut agglutinin (PNA), a plant lectin protein that recognizes the galactose ß (1 -> 3) N-acetylgalactosamine carbohydrate sequence, has been widely used as a sperm acrosome-specific marker; however, the acrosomal glycoproteins that specifically bind to PNA have yet to be identified. We herein purified and identified PNA-binding glycoproteins in the mouse testis using biotinylated PNA and streptavidin-coupled magnetic beads, and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. In six repeated experiments, sperm equatorial segment protein 1 (SPESP1) was detected most frequently as a PNA-binding glycoprotein, followed by dipeptidase 3, proacrosin-binding protein, and acrosin prepropeptide. The identification of SPEPS1 in the testis lysate and its PNA-bound fraction was verified with lectin and Western blot analyses, and the co-localization of PNA and SPEPS1 in acrosomes was confirmed with lectin- and immunohistochemistry. Since the PNA reactivity of sperm acrosomes was observed not only in normal mice, but also in SPESP1-deficient mice, although at lower levels, PNA was also considered to bind to other candidate glycoproteins. The present study identified SPESP1 in the acrosome as the primary binding target of PNA in the mouse testis. Further defining the specific lectin-glycoprotein relationships in individual cells will enhance the value of lectin histochemistry.

Autoantibody to scaffold attachment factor B (SAFB): A novel connective tissue disease-related autoantibody associated with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel connective tissue disease (CTD)-related autoantibody (autoAb) directed against scaffold attachment factor B (SAFB). METHODS: AutoAb specificity was analyzed using RNA and protein-immunoprecipitation assays. Autoimmune targets were affinity purified using patients' sera and subjected to liquid chromatography mass spectrometry. RESULTS: By immunoprecipitation assay, 10 sera reacted with a protein with a molecular weight of approximately 160 kDa. Liquid chromatography mass spectrometry of the partially purified autoantigen and additional immunoblot-based analyses revealed that the Ab specifically recognized SAFB. Anti-SAFB Abs were detected in 2 of 646 patients with systemic sclerosis (SSc) (0.3%), 1 of 1570 patients with polymyositis/dermatomyositis (0.06%), 4 of 270 patients with interstitial lung disease (ILD) (1.5%), 1 of 43 patients with overlap syndrome (2.3%) and 2 patients with other diseases including primary Raynaud's disease and eosinophilic pneumonia. Five patients with anti-SAFB Abs had Raynaud's phenomenon and 3 had nail fold punctate hemorrhage. Of note, 8 of the 10 patients (80%) suffered from ILD. None of the patients with anti-SAFB Abs had pulmonary arterial hypertension, heart disease, or renal involvement. CONCLUSIONS: Anti-SAFB Ab is a novel CTD-related autoAb possibly associated with ILD.

Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum.

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

BACKGROUND INFORMATION: Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. RESULTS: To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. CONCLUSIONS: These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels.

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.

Specific labeling of streptavidin for better understanding of ligand modification in modular method for affinity labeling (MoAL).

We studied the specific labeling of streptavidin using the modular method for affinity labeling (MoAL) that we developed based on a catalytic amide-forming reaction using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and a tertiary amine catalyst. The primary structures of avidin and streptavidin are significantly different from each other, and streptavidin does not possess an acidic amino acid equivalent to Asp108 of avidin, which is the target acidic amino acid that was labeled using MoAL. However, using biotinylated modular ligand catalysts (MLC) originally designed for labeling avidin, the labeling of streptavidin was found to successfully proceed at Glu51, which is located in a different region. The present study indicates that MoAL is readily applicable to protein labeling without a precise design for MLC. The most important factor for the design of MLC is to ensure that the linker is of sufficient length to connect the ligands to a catalytic site.

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate.

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1-0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 µg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.

Metal-coated semiconductor nanostructures and simulation of photon extraction and coupling to optical fibers for a solid-state single-photon source.

We have realized metal-coated semiconductor nanostructures for a stable and efficient single-photon source (SPS) and demonstrated improved single-photon extraction efficiency by the selection of metals and nanostructures. We demonstrate with finite-difference time-domain (FDTD) simulations that inclination of a pillar sidewall, which changes the structure to a nanocone, is effective in improving the photon extraction efficiency. We demonstrate how such nanocone structures with inclined sidewalls are fabricated with reactive ion etching. With the optimized design, a photon extraction efficiency to outer airside as high as ~97% generated from a quantum dot in a nanocone structure is simulated, which is the important step in realizing SPS on-demand operations. We have also examined the direct contact of such a metal-embedded nanocone structure with a single-mode fiber facet as a simple and practical method for preparing fiber-coupled SPS and demonstrated practical coupling efficiencies of ~16% with FDTD simulation.

Glycosylated porphyra-334 and palythine-threonine from the terrestrial cyanobacterium Nostoc commune.

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.

The defense response in Arabidopsis thaliana against Fusarium sporotrichioides.

BACKGROUND: Certain graminaceous plants such as Zea mays and Triticum aestivum serve as hosts for Fusarium sporotrichioides; however, molecular interactions between the host plants and F. sporotrichioides remain unknown. It is also not known whether any interaction between Arabidopsis thaliana and F. sporotrichioides can occur. To understand these interactions, we performed proteomic analysis. RESULTS: Arabidopsis leaves and flowers were inoculated with F. sporotrichioides. Accumulation of PLANT DEFENSIN1.2 (PDF1.2) and PATHOGENESIS RELATED1 (PR1) mRNA in Arabidopsis were increased by inoculation of F. sporotrichioides. Furthermore, mitogen-activated protein kinase 3 (MPK3) and mitogen-activated protein kinase 6 (MPK6), which represent MAP kinases in Arabidopsis, were activated by inoculation of F. sporotrichioides. Proteomic analysis revealed that some defense-related proteins were upregulated, while the expression of photosynthesis- and metabolism-related proteins was down regulated, by inoculation with F. sporotrichioides. We carried out the proteomic analysis about upregulated proteins by inoculation with Fusarium graminearum. The glutathione S-transferases (GSTs), such as GSTF4 and GSTF7 were upregulated, by inoculation with F. graminearum-infected Arabidopsis leaves. On the other hand, GSTF3 and GSTF9 were uniquely upregulated, by inoculation with F. sporotrichioides. CONCLUSIONS: These results indicate that Arabidopsis is a host plant for F. sporotrichioides. We revealed that defense response of Arabidopsis is initiated by infection with F. sporotrichioides.

Proteomic analysis of S-nitrosylation induced by 1-methyl-4-phenylpyridinium (MPP+).

BACKGROUND: Nitric oxide (NO) mediates its function through the direct modification of various cellular targets. S-nitrosylation is a post-translational modification of cysteine residues by NO that regulates protein function. Recently, an imbalance of S-nitrosylation has also been linked to neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. RESULTS: In the present study, we used two-dimensional gel electrophoresis in conjunction with the modified biotin switch assay for protein S-nitrosothiols using resin-assisted capture (SNO-RAC) to identify proteins that are S-nitrosylated more intensively in neuroblastoma cells treated with a mitochondrial complex I inhibitor, 1-methyl-4-phenylpyridinium (MPP+). We identified 14 proteins for which S-nitrosylation was upregulated and seven proteins for which it was downregulated in MPP+-treated neuroblastoma cells. Immunoblot analysis following SNO-RAC confirmed a large increase in the S-nitrosylation of esterase D (ESD), serine-threonine kinase receptor-associated protein (STRAP) and T-complex protein 1 subunit γ (TCP-1 γ) in MPP+-treated neuroblastoma cells, whereas S-nitrosylation of thioredoxin domain-containing protein 5 precursor (ERp46) was decreased. CONCLUSIONS: These results suggest that S-nitrosylation resulting from mitochondrial dysfunction can compromise neuronal survival through altering multiple signal transduction pathways and might be a potential therapeutic target for neurodegenerative diseases.

Gene expression analysis of wounding-induced root-to-shoot communication in Arabidopsis thaliana.

Root-to-shoot communication plays an important role in the adaptation to environmental stress. In this study, we established a model system for root-to-shoot signalling to observe global gene expression in Arabidopsis thaliana. The roots of Arabidopsis seedlings were wounded and the expression in the shoots of 68 and 5 genes was up-regulated threefold at 30 min and 6 h post-injury, respectively. These genes were designated early and late Root-to-Shoot responsive (RtS) genes, respectively. Many of the early RtS genes were found to encode transcription factors such as AtERFs, whereas others were associated with jasmonic acid (JA) and ethylene (ET). Some of the late RtS genes were shown to be regulated by 12-oxo-phytodienoic acid (OPDA). In fact, elevated levels of JA and OPDA were detected in the shoots of seedlings 30 min and 6 h, respectively, after wounding of the roots. A mutant analysis revealed that JA and ET are involved in the expression of the early RtS genes. Thus, root-to-shoot communication for many RtS genes is associated with the systemic production of JA, OPDA and possibly ET.

Plant cells without detectable plastids are generated in the crumpled leaf mutant of Arabidopsis thaliana.

Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. It is unclear, however, whether cells without plastids are generated when plastid division is suppressed. The crumpled leaf (crl) mutant of Arabidopsis thaliana is a plastid division mutant that displays severe abnormalities in plastid division and plant development. We show that the crl mutant contains cells lacking detectable plastids; this situation probably results from an unequal partitioning of plastids to each daughter cell. Our results suggest that crl has a partial defect in plastid expansion, which is suggested to be important in the partitioning of plastids to daughter cells when plastid division is suppressed. The absence of cells without detectable plastids in the accumulation and replication of chloroplasts 6 (arc6) mutant, another plastid division mutant of A. thaliana having no significant defects in plant morphology, suggests that the generation of cells without detectable plastids is one of the causes of the developmental abnormalities seen in crl plants. We also demonstrate that plastids with trace or undetectable amounts of chlorophyll are generated from enlarged plastids by a non-binary fission mode of plastid replication in both crl and arc6.

Characterization of extracellular matrix components from the desiccation-tolerant cyanobacterium Nostoc commune.

The terrestrial cyanobacterium Nostoc commune forms macroscopic colonies in its natural habitats, and these colonies consist of both cellular filaments and massive extracellular matrixes. In this study, the biochemical features of the extracellular matrix components were investigated. Naturally growing N. commune was tolerant to desiccation, and produced massive extracellular polysaccharides that contained both neutral sugars and glucuronic acid as constituent monosaccharides. The extracellular polysaccharide contents and desiccation tolerance were compared in laboratory culture strains of Nostoc species. The laboratory culture of N. commune strain KU002 was sensitive to desiccation and produced smaller amounts of extracellular polysaccharides, unlike the field-isolated naturally growing colonies. Nostoc punctiforme strain M-15, which is genetically closed to N. commune, was able to tolerate desiccation, although the other Nostoc strains were desiccation-sensitive. A laboratory culture strain of the aquatic cyanobacterium Nostoc sphaericum produced massive extracellular polysaccharides but was sensitive to desiccation, suggesting that extracellular matrix production is not enough to make this strain tolerant to desiccation. WspA (water stress protein) and SodF (superoxide dismutase) were found to be characteristic protein components of the extracellular matrix of N. commune. Because the WspA proteins were heterogeneous, the wspA genes were highly diverse among the different genotypes of N. commune, although the sodF gene was rather conservative. The heterogeneity of the WspA proteins suggests their complex roles in the environmental adaptation mechanism in N. commune.

Characterization of mycosporine-like amino acids in the cyanobacterium Nostoc verrucosum.

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for >90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.

Cerebrospinal fluid biomarkers showing neurodegeneration in dogs with GM1 gangliosidosis: possible use for assessment of a therapeutic regimen.

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.

Characterization of the chemical diversity of glycosylated mycosporine-like amino acids in the terrestrial cyanobacterium Nostoc commune.

Mycosporine-like amino acids (MAAs) are UV-absorbing pigments, and structurally unique glycosylated MAAs are found in the terrestrial cyanobacterium Nostoc commune. In this study, we examined two genotypes of N.commune colonies with different water extract UV-absorption spectra. We found structurally distinct MAAs in each genotype. The water extract from genotype A showed a UV-absorbing spectrum with an absorption maximum at 335nm. The extract contained the following compounds: 7-O-(ß-arabinopyranosyl)-porphyra-334 (478Da), pentose-bound shinorine (464Da), hexose-bound porphyra-334 (508Da) and porphyra-334 (346Da). The water extract from genotype B showed a characteristic UV-absorbing spectrum with double absorption maxima at 312 and 340nm. The extract contained hybrid MAAs (1050Da and 880Da) with two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen linked to 2-O-(ß-xylopyranosyl)-ß-galactopyranoside. A novel 273-Da MAA with an absorption maximum at 310nm was also identified in genotype B. The MAA consisted of a 3-aminocyclohexen-1-one linked to a γ-aminobutyric acid chain. These MAAs had potent radical scavenging activities in vitro and the results confirmed that the MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune. The two genotypes of N. commune exclusively produced their own characteristic glycosylated MAAs, which supports that MAA composition could be a chemotaxonomic marker for the classification of N. commune.