Clinical Course of Atopic Dermatitis in an Adult with Amyotrophic Lateral Sclerosis: Aetiological Implications of Voluntary Movements and Dermatitis Severity.

is missing (Short communication).

[Progressive multifocal leukoencephalopathy during the treatment for mycosis fungoides].

A 58-year-old man was diagnosed with mycosis fungoides (MF) confirmed by skin biopsy for systemic erythema that appeared in 2006 and had been on psoralen plus ultraviolet A (PUVA) therapy and topical steroids. In September 2017, he had diffuse large B-cell lymphoma and received chemotherapy. Since March 2019, tumor stage MF with large cell transformation was observed, and chemotherapy containing brentuximab vedotin (BV) was performed, which yielded a remarkable response. During the preparation for allogeneic hematopoietic stem cell transplantation, bradykinesia, delayed response, and cognitive decline were observed. Head magnetic resonance imaging fluid-attenuated inversion recovery images showed hyperintensity in the deep white matter below the bilateral frontal cortex. The general cerebrospinal fluid test revealed no abnormalities and was below the sensitivity of JC virus (JCV) quantitative PCR. As progressive multifocal leukoencephalopathy (PML) was strongly suspected from clinical symptoms and radiographic signs, ultrasensitive JCV testing was performed. The test result was positive; hence, the patient was diagnosed with PML. Chemotherapy was discontinued, but his central nervous system symptoms worsened, and he died on the 135th day of illness. We considered that PML developed based on the underlying disease and immunodeficiency caused by chemotherapy such as BV.

Insight into innate immune response in "Yusho": The impact of natural killer cell and regulatory T cell on inflammatory prone diathesis of Yusho patients.

BACKGROUND: In 1968 in western Japan, polychlorinated biphenyl-contaminated "Kanemi rice oil" was used in cooking, causing food poisoning in many people. More than 50 years have passed since the Yusho incident, and although inflammatory disorders such as suppuration have been observed in Yusho patients, the etiology of this inflammation susceptibility remains obscure. OBJECTIVES: To investigate the mechanisms of susceptibility to inflammation in Yusho patients, peripheral immune cell fractions and concentrations of inflammatory cytokines were evaluated in blood samples collected from both Yusho patients and age-matched healthy subjects undergoing medical examination in Nagasaki. METHODS: To exclude diagnostic uncertainty, serum levels of polychlorinated biphenyl (PCB), polychlorinated quarterphenyl (PCQ), and polychlorinated dibenzofuran (PCDF) were measured. Immune cell (e.g. natural killer and regulatory T cell) populations were analyzed by flow cytometry. Serum cytokines involved in immune cell activation were measured by ELISA. RESULTS: The relative proportion of natural killer cells was higher in Yusho patients than in healthy subjects, while the proportion of regulatory T cells did not differ between groups. Serum concentrations of IL-36 and IFN-γ were significantly lower in Yusho patients than in healthy subjects. Conversely, serum cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which is a cytokine related to activated NK cells, was higher in Yusho patients than in healthy subjects and was positively correlated with PCDF blood levels. CONCLUSION: Increased numbers of NK cells in Yusho patients suggests that the innate immune response has been activated in Yusho patients. The seemingly paradoxical results for CTLA-4 and IFN-γ may reflect counterbalancing mechanisms preventing excessive NK cell activation. This dysregulation of innate immunity might contribute to the inflammation observed in Yusho patients.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers.

Polyamine oxidase (PAO), which requires FAD as a cofactor, functions in polyamine catabolism. Plant PAOs are classified into two groups based on their reaction modes. The terminal catabolism (TC) reaction always produces 1,3-diaminopropane (DAP), H2O2, and the respective aldehydes, while the back-conversion (BC) reaction produces spermidine (Spd) from tetraamines, spermine (Spm) and thermospermine (T-Spm) and/or putrescine from Spd, along with 3-aminopropanal and H2O2. The Oryza sativa genome contains seven PAO-encoded genes termed OsPAO1-OsPAO7. To date, we have characterized four OsPAO genes. The products of these genes, i.e. OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze BC-type reactions. Whereas OsPAO1 remains in the cytoplasm, the other three PAOs localize to peroxisomes. Here, we examined OsPAO7 and its gene product. OsPAO7 shows high identity to maize ZmPAO1, the best characterized plant PAO having TC-type activity. OsPAO7 seems to remain in a peripheral layer of the plant cell with the aid of its predicted signal peptide and transmembrane domain. Recombinant OsPAO7 prefers Spm and Spd as substrates, and it produces DAP from both substrates in a time-dependent manner, indicating that OsPAO7 is the first TC-type enzyme identified in O. sativa. The results clearly show that two types of PAOs co-exist in O. sativa. Furthermore, OsPAO7 is specifically expressed in anthers, with an expressional peak at the bicellular pollen stage. The physiological function of OsPAO7 in anthers is discussed.

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the ß-barrel pore formation mechanism by two components.

Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a ß-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting ß2 and ß3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal ß-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a ß-sheet in the monomer (prestem), and interaction with the adjacent protomer.

Preliminary X-ray crystallographic study of staphylococcal α-haemolysin monomer.

Staphylococcal α-haemolysin is a ß-barrel pore-forming toxin expressed by Staphylococcus aureus. α-Haemolysin is secreted as a water-soluble monomeric protein which binds to target membranes and forms membrane-inserted heptameric pores. Although the crystal structures of the heptameric pore and monomer bound to an antibody have been determined, that of monomeric α-haemolysin without binder has yet to be elucidated. Previous mutation studies showed that mutants of His35 retain the monomeric structure but are unable to assemble into heptamers. Here, α-haemolysin H35A mutants were expressed, purified and crystallized. Diffraction data were collected to 2.90 Å resolution. The crystals belonged to space group P61, with unit-cell parameters a = b = 151.3, c = 145.0 Å. Molecular replacement found four molecules in an asymmetric unit. The relative orientation among molecules was distinct from that of the pore, indicating that the crystal contained monomeric α-haemolysin.

AxyR, an AraC family transcriptional activator of the xylanase 3 gene, requires xylo-oligosaccharides as a cofactor for DNA binding in Paenibacillus sp. strain W-61.

The xylanolytic bacterium Paenibacillus sp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3 gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3 promoter region.

Cadaverine covalently linked to the peptidoglycan serves as the correct constituent for the anchoring mechanism between the outer membrane and peptidoglycan in Selenomonas ruminantium.

In Selenomonas ruminantium, a strictly anaerobic and gram-negative bacterium, cadaverine covalently linked to the peptidoglycan is required for the interaction between the peptidoglycan and the S-layer homologous (SLH) domain of the major outer membrane protein Mep45. Here, using a series of diamines with a general structure of NH(3)(+)(CH(2))(n)NH(3)(+) (n = 3 to 6), we found that cadaverine (n = 5) specifically serves as the most efficient constituent of the peptidoglycan in acquiring the high resistance of the cell to external damage agents and is required for effective interaction between the SLH domain of Mep45 and the peptidoglycan, facilitating the correct anchoring of the outer membrane to the peptidoglycan.

Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal ß-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal ß-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded ß-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

Cell surface xylanases of the glycoside hydrolase family 10 are essential for xylan utilization by Paenibacillus sp. W-61 as generators of xylo-oligosaccharide inducers for the xylanase genes.

Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Deltaxyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5DeltaSLH) grew poorly, secreting Xyn5DeltaSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system.

The membrane lipoprotein LppX of Paenibacillus sp. strain W-61 serves as a molecular chaperone for xylanase of glycoside hydrolase family 11 during secretion across the cytoplasmic membrane.

Paenibacillus sp. strain W-61, which can utilize xylan as the sole source of carbon and energy, produces extracellular xylanases 1 and 3 (Xyn1 and Xyn3) and cell surface xylanase 5. In this study we found that lppX, immediately downstream of xyn1, encodes a lipoprotein located on the outer layer of the cytoplasmic membrane and that the LppX lipoprotein is essential for the secretion of active Xyn1 across the cytoplasmic membranes. In Escherichia coli, wild-type LppX was destined for the inner layer of the outer membrane. Mutant LppX(C19A), in which Cys-19, a possible lipomodification residue, is replaced with Ala, was located in the periplasm without being anchored to the membranes. Another mutant, LppX(S20D S21D), with substitutions of Asp for Ser-20 and Ser-21 (conversion to an Asp-Asp signal for sorting to the inner membrane), resided on the outer layer of the inner membrane, demonstrating that LppX has the sorting property of a lipoprotein. E. coli harboring both xyn1 and lppX secreted active Xyn1 into the periplasm. In contrast, E. coli carrying xyn1 alone failed to do so, accumulating inactive Xyn1 in the cytoplasmic membranes. Exogenous LppX(C19A) liberated the inactive Xyn1, which had been stagnating in the inner membrane, into the medium as an active enzyme. Thus, we propose that LppX is a novel type of lipoprotein that assists Xyn1 in making the proper fold necessary for traveling across the cytoplasmic membranes to be secreted as an active enzyme.

Identification of ORF636 in phage phiSLT carrying Panton-Valentine leukocidin genes, acting as an adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of Staphylococcus aureus.

The temperate phage phiSLT of Staphylococcus aureus carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of S. aureus. ORF636 bound specifically to S. aureus; it did not bind to any other staphylococcal species or to several gram-positive bacteria.

Two segments in bacterial antizyme P22 are essential for binding and enhance degradation of lysine/ornithine decarboxylase in Selenomonas ruminantium.

In Selenomonas ruminantium, a strictly anaerobic and gram-negative bacterium, the degradation of lysine/ornithine decarboxylase (LDC/ODC) by ATP-requiring protease(s) is accelerated by the binding of P22, which is a ribosomal protein of this strain. Amino acid sequence alignment of S. ruminantium P22 with the L10 ribosomal proteins of gram-positive and -negative bacteria showed that P22 has a 5-residue K101NKLD105 segment and an 11-residue G160VIRNAVYVLD170 segment, both of which are lacking in L10 in any other gram-positive and gram-negative bacteria reported. To elucidate whether the two segments are involved in P22 function, a series of mutant genes of P22 were constructed and expressed in Escherichia coli. The proteins were isolated and assayed for their function with respect to S. ruminantium LDC/ODC and mouse ODC. The results indicated that the two segments of P22 are crucial for P22 binding to both enzymes and also accelerated degradation of both decarboxylases.

Binding sequences for RdgB, a DNA damage-responsive transcriptional activator, and temperature-dependent expression of bacteriocin and pectin lyase genes in Pectobacterium carotovorum subsp. carotovorum.

Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23 degrees C) differs from that for synthesis of Pnl (30 degrees C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P(0), P(1), and P(2) promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K(d) [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K(d) = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23 degrees C compared with that at 30 degrees C. In contrast, the amount of pnl transcription tripled at 30 degrees C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30 degrees C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

Cloning, expression, and transglycosylation reaction of Paenibacillus sp. strain W-61 xylanase 1.

A xylanase gene, xyn1, which encodes Paenibacillus sp. strain W-61 xylanase 1 (Xyn1), was cloned in Escherichia coli. xyn1 encodes 211 amino acid residues, including 28 amino acid residues of a signal peptide. The deduced amino acid sequence of the mature Xyn1 showed 95.7%, 84.0%, and 83.7% identity to family 11 xylanases of Aeromonas caviae ME-1, Paenibacillus sp., and Bacillus stearothermophilus respectively. The physico-chemical properties of recombinant Xyn1 were very similar to those of intact Xyn1, except for the molecular mass. The pattern of xylooligosaccharides generated by rXyn1 was investigated by fluorophore-assisted carbohydrate electrophoresis (FACE). The degradation rate of xylohexaose by rXyn1 increased markedly as compared with that of xylopentaose. Xylohexaose had a single preferential point of cleavage by rXyn1. On the basis of the pattern of action of xylooligosaccharides, the number of subsites was estimated to be six. The catalytic site was located between the third and the fourth subsites from non-reducing end.

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium.

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.

Expression of gamma-hemolysin regulated by sae in Staphylococcus aureus strain Smith 5R.

Staphylococcus aureus strain Smith 5R produces a two-component pore-forming toxin and forms a rough-surfaced colony with hemolytic haloes on human red blood cell plates (R[+]). Serial subcultures of the strain in broth caused the appearance of gamma-hemolysin negative variants with a smooth colony shape (S[-]), and the S[-] valiant became predominant in culture. The R[+] strain, in which agrA is naturally disrupted by an insertion of IS1181, produced high levels of gamma-hemolysin. In the S[-] variant, expression of both hlg and lukS-F mRNAs was strongly reduced. Nucleotide sequencing of the sae locus revealed that all isolated S[-] variants had spontaneous mutations in the sae locus. Recovery of gamma-hemolysin productivity in S[-] by transformation of the wild-type sae allele strongly suggested that the expression of gamma-hemolysin is positively regulated by sae in an agr-independent manner.

Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus.

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Here, we cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. The genomic regions encoding pleurotolysin A and B contained two and eight introns, respectively, and putative promoter sequences. The complementary DNA (cDNA) for pleurotolysin A encoded 138 amino acid residues, and the predicted product was identical with natural pleurotolysin A, except for the presence of the first methionine. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively, which were unfolded with 8 M urea and refolded by 100-fold dilution with 10 mM Tris-HCl buffer, pH 8.5. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell as efficiently as the natural proteins at nanomolar concentrations. In contrast, the precursor of pleurotolysin B was much less hemolytically active than mature pleurotolysin B in the presence of pleurotolysin A.

The association between Staphylococcus aureus strains carrying panton-valentine leukocidin genes and the development of deep-seated follicular infection.

BACKGROUND: Panton-Valentine leukocidin (PVL) is mainly associated with necrotic suppurative lesions, such as furuncles and abscesses in the skin and subcutaneous tissue, but it has also been isolated from patients with community-acquired, severe, necrotizing pneumonia. However, the clinical manifestations of furuncles caused by PVL-producing Staphylococcus aureus and the role of patients' background are not fully understood. METHODS: We used polymerase chain reaction amplification to test for the PVL gene in 161 strains of S. aureus isolated from suppurative skin lesions. For all PVL gene-positive strains isolated from furuncles, we analyzed cutaneous manifestations, patient background characteristics, and bacteriological markers, including coagulase types, presence of the mecA gene, and toxin profiles, and we compared these results with those for PVL gene-negative strains. RESULTS: PVL genes were detected in 16 (40%) of the 40 S. aureus strains isolated from furuncles, 2 (28%) of the 7 strains isolated from carbuncles, 1 (14%) of the 7 strains isolated from abscesses, and 1 (5%) of the 20 strains isolated from folliculitis. PVL gene-positive S. aureus usually causes multiple (rather than single) furuncles, and such furuncles are usually associated with more-intense erythema around the lesions. PVL gene-positive strains were isolated from young adults without underlying diseases, whereas PVL gene-negative strains were isolated from patients with various systemic complications, including diabetes, leukemia, and autoimmune diseases. CONCLUSIONS: PVL gene-positive S. aureus strains are involved in the development of multiple furuncles with more-intense erythema, particularly in healthy young adults. An understanding of the characteristics of furuncles due to PVL gene-positive strains might be useful for preventing the development of the severe systemic infections.