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Vitamin C (VitC) is maintained at high concentrations in the brain and is an essential micronutrient for brain function. VitC deficiency leads to neuropsychiatric scurvy, which is characterized by depression and cognitive impairment. However, the molecular mechanism by which mild VitC deficiency impairs brain function is currently unknown. In the present study, we conducted RNA sequencing analysis and found that a short-term VitC deficiency altered the brain transcriptome in ODS rats, which cannot synthesize VitC. Bioinformatic analysis indicated that VitC deficiency affected the expression of genes controlled by the glucocorticoid receptor in the brain. We confirmed an increased secretion of glucocorticoids from the adrenal gland during VitC deficiency. We found that non-neuronal cells, including microglia, which are resident immune cells in the brain, changed their transcriptional patterns in response to VitC deficiency. Immunohistochemical analysis revealed that the quiescent ramified microglia transform into the activated amoeboid microglia during three weeks of VitC deficiency. The morphological activation of microglia was accompanied by increased expression of proinflammatory cytokines such as interleukin-6 in the hippocampus. Furthermore, VitC deficiency decreased the number of newly born neurons in the dentate gyrus of the hippocampus, suggesting that VitC was required for adult neurogenesis that plays a crucial role in learning and memory. Our findings may provide insights into the molecular mechanisms underlying the maintenance of normal brain function by adequate levels of VitC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that may have influenced the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a β-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180 g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Zizania latifolia cultivars infected by the endophytic fungus Ustilago esculenta develop an edible stem gall. Stem gall development varies among cultivars and individuals and may be affected by the strain of U. esculenta. To isolate haploids from two Z. latifolia cultivars in our paddy fields, Shirakawa and Ittenkou, we herein performed the sporadic isolation of U. esculenta strains from stem gall tissue, a PCR-based assessment of the mating type, and in vitro mating experiments. As a result, we obtained heterogametic strains of MAT-2 and MAT-3 as well as MAT-2, but not MAT-3, haploid strains. Another isolation method, in which we examined poorly growing small clusters of sporidia derived from teliospores, succeeded in isolating a MAT-3 haploid strain. We also identified the mating types of 10 U. esculenta strains collected as genetic resources from different areas in Japan. All strains, except for one MAT-1 haploid strain, were classified as MAT-2 haploid strains or heterogametic strains of MAT-2 and MAT-3. The isolated strains of MAT-1, MAT-2, and MAT-3 mated with each other to produce hyphae. Collectively, these results indicate that the mating types of U. esculenta infecting Z. latifolia cultivars in Japan are biased towards MAT-2 and MAT-3 and that U. esculenta populations in these Japanese cultivars may be characterized by the low isolation efficiency of the MAT-3 haploid.

An α-glucosidase from Aspergillus sojae, AsojAgdL, exhibits strong transglucosylation activity to produce α-1,6-glucosidic linkages. The most remarkable structural feature of AsojAgdL is that residues 457-560 of AsojAgdL (designated the NC sequence) is not conserved in other glycoside hydrolase family 31 enzymes, and part of this NC sequence is proteolytically cleaved during its maturation. In this study, the enzyme was expressed in Pichia pastoris, and electrophoretic analysis indicated that the recombinant enzyme, rAsojAgdL, consisted of two polypeptide chains, as observed in the case of the enzyme produced in an Aspergillus strain. The crystal structure of rAsojAgdL was determined in complex with the substrate analog trehalose. Electron density corresponding to residues 496-515 of the NC sequence was not seen, and there were no α-helices or β-strands except for a short α-helix in the structures of residues 457-495 and residues 516-560, both of which belong to the NC sequence. The residues 457-495 and the residues 516-560 both formed extra components of the catalytic domain. The residues 457-495 constituted the entrance of the catalytic pocket of rAsojAgdL, and Gly467, Asp468, Pro469, and Pro470 in the NC sequence were located within 4 Å of Trp400, a key residue involved in binding of the substrate. The results suggest that the proteolytic processing of the NC sequence is related to the formation of the catalytic pocket of AsojAgdL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF 2 ), tetrasaccharide nystose (GF 3 ), and fructosyl nystose (GF 4 ). Enzymes that hydrolyze GF 3 may be useful for preparing GF 2 from the fructooligosaccharide mixture. A β-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60 mM sucrose, GF 2 , and GF 3 by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF 2 was lower than those for disaccharide sucrose and tetrasaccharide GF 3 . The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial β-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF 2 and the amino acid residues interacting with fructose at subsite - 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60 mM GF 2 /GF 3 by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117-127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF 2 . KEY POINTS: • Frischella β-fructofuranosidase hydrolyzed nystose more efficiently than 1-kestose. • Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference. • Loop consisting of residues 117-127 appears to contribute to the substrate binding., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the long-distance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement., (Copyright © 2021. Published by Elsevier B.V.)

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales . Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance., (© 2021. The Author(s).)

Background: Recently, the relationship between antigen contact via skin (skin sensitization) and the development of food allergies has gained increasing attention. However, few studies have examined the effects of skin sensitization on healthy skin., Objective: To examine the effect of sensitization in healthy skin on IgE and cytokine production during food allergy development., Methods: The effect of skin sensitization on food allergy was evaluated using DO11.10 mice whose T cells express ovalbumin (OVA)-specific T-cell receptors. OVA was applied to the back skin of mice dehaired by various methods, and then food allergy was induced by providing them with an OVA-containing diet. OVA-specific IgE production in the sera and decreases in body temperature due to anaphylactic reaction were measured as indicators of food allergy. In addition, IL-4 production and proliferation of splenocytes were measured in mice with food allergy after skin sensitization., Results: Skin sensitization in healthy skin increased IgE production and exacerbated anaphylactic symptoms induced by ingesting the antigen. Moreover, skin sensitization enhanced IL-4 production from splenocytes during the onset of food allergy. In contrast, oral tolerance was induced even after establishing skin sensitization., Conclusion: Skin sensitization temporarily exacerbated food allergy by enhancing systemic Th2 responses. These findings will help identify the mechanisms involved in food allergy and help develop treatments., (© 2020 S. Karger AG, Basel.)

Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA viruses to determine their host ranges. Many viruses including Tomato mosaic virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the 32 P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with virus cell-to-cell movement.

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement.

The root diameters as well as colonization and diversity of the root-associating fungi of Vaccinium oldhamii Miq. were investigated in order to obtain information on their mycorrhizal properties. The distal regions of roots had typical hair roots with diameters of less than 100 μm. Ericoid mycorrhizal fungi (ErMF) and dark septate endophytes (DSE) were frequently observed in the roots. Ascomycetes, particularly helotialean fungi, appeared to be dominant among the endophytic fungi of V. oldhamii roots. Furthermore, Rhizoscyphus ericae (Read) Zhuang & Korf and Oidiodendron maius Barron known as ErMF were detected more frequently than other fungal species.

To adapt to plants as hosts, plant viruses have evolutionally needed the capacity to modify the host plasmodesmata (PD) that connect adjacent cells. Plant viruses have acquired one or more genes that encode movement proteins (MPs), which facilitate the cell-to-cell movement of infectious virus entities through PD to adjacent cells. Because of the diversity in their genome organization and in their coding sequences, rice viruses may each have a distinct cell-to-cell movement strategy. The complexity of their unusual genome organizations and replication strategies has so far hampered reverse genetic research on their genome in efforts to investigate virally encoded proteins that are involved in viral movement. However, the MP of a particular virus can complement defects in cell-to-cell movement of other distantly related or even unrelated viruses. Trans-complementation experiments using a combination of a movement-defective virus and viral proteins of interest to identify MPs of several rice viruses have recently been successful. In this article, we reviewed recent research that has advanced our understanding of cell-to-cell movement of rice viruses.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His(6)-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

The nonstructural protein pC6 encoded by rice grassy stunt virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe virus, which can support viral cell-to-cell movement. In a trans-complementation experiment with a movement-defective tomato mosaic virus, pC6 and pC4 facilitated intercellular transport of the virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement.

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.

Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.

Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.

A hybrid Cowpea chlorotic mottle virus(CCMV) with the movement protein (MP) gene replaced with that of the closely related Brome mosaic virus cannot infect cowpea systemically. Twenty-nine spontaneous mutants from the hybrid CCMV capable of systemic infection in cowpea appeared through biased codon changes that resulted in Lys or Arg at five specific positions in the MP gene. In this study, we report that systemic infection of cowpea with the hybrid CCMV can be achieved by artificial codon changes that do not result in Lys or Arg. We discuss mechanisms that restrict the occurrence of cowpea-adapted mutants in nature.

Background/aims: Conventional methods predicting survival in patients with primary biliary cirrhosis are based on the results of blood tests and on clinical condition, both of which may be affected by treatment. Portal circulation can be evaluated in a relatively noninvasive manner by per-rectal portal scintigraphy. We used this method to evaluate portal hemodynamics and assess prognosis in patients with primary biliary cirrhosis., Methodology: Per-rectal portal scintigraphy with Tc-99m pertechnetate was done in 51 patients with primary biliary cirrhosis. A solution containing Tc-99m pertechnetate was instilled into the rectum, and serial scintigrams were taken while radioactivity curves for the liver and heart were recorded sequentially. The per-rectal portal shunt index was calculated from the curves., Results: The shunt index was higher in patients with stage IV primary biliary cirrhosis than in those with stage I, II, or III primary biliary cirrhosis. On the basis of portal shunt index, the patients were divided into those with a shunt index of less than 18%, and those with a shunt index of 18% or more. The cumulative survival rate was lower among patients with the higher shunt index. On regression analysis, the portal shunt index was found to be significantly related to survival., Conclusions: Our results indicate that per-rectal portal scintigraphy with Tc-99m pertechnetate can be used to non-invasively evaluate the portal circulation of patients with primary biliary cirrhosis and is useful in establishing prognosis in such patients.

Background/aims: Portal circulation can be evaluated in a relatively noninvasive manner by per-rectal portal scintigraphy. We used this method to evaluate portal hemodynamics in patients with primary biliary cirrhosis and idiopathic portal hypertension. We did the procedures simultaneously in some patients to examine the relation between portal circulation and hepatic functional reserve in these diseases., Methodology: Per-rectal portal scintigraphy with Tc-99m pertechnetate was done in 17 healthy subjects, 154 patients with chronic hepatitis, 447 patients with cirrhosis, 40 patients with primary biliary cirrhosis, and 20 patients with idiopathic portal hypertension. Eighty-three patients (14 with hepatitis, 48 with cirrhosis, 16 with primary biliary cirrhosis, and 5 with idiopathic portal hypertension) also underwent scintigraphy with Tc-99m galactosyl human serum albumin with 2 weeks. A solution containing Tc-99m pertechnetate was instilled into the rectum, and serial scintigrams were taken while radioactivity curves for the liver and heart were recorded sequentially. The per-rectal portal shunt index was calculated from the curves. A receptor index was calculated by dividing the radioactivity of the liver region of interest by that of the liver-plus-heart region of interest 15 min after the injection of Tc-99m galactosyl human serum albumin. The index of blood clearance was calculated by dividing the radioactivity of the heart region of interest at 15 min by that of the heart region of interest at 3 min., Results: The shunt index was higher for more severe disorders, increasing in the order of chronic hepatitis, cirrhosis without varices, and cirrhosis with varices. The shunt indices in patients with primary biliary cirrhosis and idiopathic portal hypertension were higher than that in patients with chronic hepatitis. In terms of receptor index, the standard residuals were more than 0 in 10 of 16 patients with primary biliary cirrhosis and 4 of 5 patients with idiopathic portal hypertension. In terms of index of blood clearance, the standard residuals were more than 0 in 10 of 16 patients with primary biliary cirrhosis and 4 of 5 patients with idiopathic portal hypertension, Conclusions: Abnormalities of portal hemodynamics in patients with primary biliary cirrhosis or idiopathic portal hypertension occur while hepatic functional reserve is still satisfactory as compared with patients who have chronic hepatitis or cirrhosis.

We examined the effects of interferon-alpha or -beta therapy on gastric emptying and digestive symptoms. The effects of cisapride on gastric emptying and digestive symptoms were also evaluated. The subjects were 48 patients with chronic hepatitis C. All patients were randomly assigned to one of four groups (A, interferon-alpha group; B, interferon-alpha and cisapride group; C, interferon-beta group; D, interferon-beta and cisapride group). Gastric emptying was measured before initiation of interferon therapy and two weeks after initiation of therapy. The half-time of gastric emptying (T1/2) was calculated. The T1/2 ratio was calculated by dividing the T1/2 after interferon therapy by the T1/2 before interferon therapy. Digestive symptom scores were determined at the time of the gastric emptying tests. The T1/2 after interferon therapy was higher than that before therapy in groups A and C (P = 0.002 and 0.059, respectively). The digestive symptom score after interferon therapy was higher than that before therapy in groups A and C (P = 0.012 and 0.093, respectively). The T1/2 ratio in group B was significantly lower than that in group A (P = 0.021), and the T1/2 ratio in group D was lower than that in group C, but the difference did not reach statistical significance (P = 0.057). Interferon-alpha is associated with a greater delay in gastric emptying and a higher symptom score than is interferon-beta. Administration of cisapride corrects the delayed gastric emptying and relieves associated digestive symptoms.