Molecular phylogeny of Punctaria mageshimensis reveals evidence for its transfer to Spatoglossum as S. mageshimense (Dictyotales,Phaeophyceae)

Taxonomy of the little‐studied brown algal species Punctaria mageshimensis (Ectocarpales s.l.) was reexamined by molecular phylogeny and morphology. In the genetic analyses of newly collected specimens using plastid rbcL and psaA gene sequences, the specimens morphologically referable to P. mageshimensis were phylogenetically distant from Ectocarpales s.l. and were included in the clade of Spatoglossum (Dictyotales). Morphological reexamination of the type specimen and newly collected specimens confirmed its systematic position in Dictyotales: Branched thallus; cushion‐shaped rhizoidal holdfast occasionally forming secondary holdfast at the bottom of the thallus; many discoidal plastids without pyrenoid per cell; tetrasporangium‐like reproductive structures with dark, homogeneous cell content; occurrence of hair tufts. Genetically P. mageshimensis was most related to a reported sequence of Spatoglossum asperum, but P. mageshimensis was considerably different from S. asperum as well as other known Spatoglossum species in the deep habitat and in having scarcely‐branched lanceolate and considerably thickened thallus. In conclusion, we propose the transfer of P. mageshimensis to Spatoglossum as S. mageshimense comb. nov.     

Sulfurimicrobium lacus gen. nov., sp. nov., a sulfur oxidizer isolated from lake water,and review of the family Sulfuricellaceae to show that it is not a later synonym of Gallionellaceae

Archives of Microbiology - A facultatively anaerobic sulfur-oxidizing bacterium, strain skT11T, was isolated from anoxic lake water of a stratified freshwater lake. As electron donor for...     

Time constraints related to sexual maturation and prolonged copulation in the female‐dimorphic damselfly Ischnura senegalensis

Time constraints are critical for reproductive success. To understand the spatiotemporal dynamics of morph frequency in the female‐dimorphic damselfly Ischnura senegalensis, we compared two different morphs for two important time constraints on female reproductive output, i.e. post‐emergence sexual maturation and prolonged copulation. The females of both morphs achieved sexual maturation 4–5 days after emergence, suggesting that the rate of sexual mutation does not result in morph‐specific fitness. The copulation durations declined with the time of onset of copulation in both morphs. Consequently, all copulations terminated at approximately 12:00 hours. Because females show foraging and oviposition activity only after copulation, the copulation duration does not result in morph‐specific time constraints. These two important time constraints do not account for morph‐specific reproductive success and do not affect the evolutionary equilibrium of morph frequency in I. senegalensis.     

Irreversible inactivation of glutathione S-transferase-π by a low concentration of naphthoquinones

π-Class glutathione S-transferase (GST-π) was very potently inactivated by oxidants such as H₂O₂, xanthine-xanthine oxidase and naphthoquinones. Thiols and glutathione analogs including dithiothreitol, reduced gluta-thione, cysteine, cysteamine, S-methyl-SG, S-hexyl-SG and S-decyl-SG protected GST-π from the inactivation, but a substrate analog (2,4-dinitrophenol), superoxide dismutase and catalase did not, suggesting that the cysteinyl residue(s) in/nearby the glutathione binding site (G-site) may be oxidatively modified by these oxidants. Many reductants and radical scavengers including butylated hydoxytoluene, α-tocopherol, ascorbate, uric acid, mannitol, tyrosine, tryptophan, histidine, quercitrin and bilirubin had no effect on the inactivation. GST-π pretreated with cystamine was reactivated very efficiently by 50 mM DTT following incubation with 1,2-naphthoquinone, whereas cystamine-untreated GST-π was not reactivated.     

Pifithrin-μ, an Inhibitor of Heat-Shock Protein 70, Can Increase the Antitumor Effects of Hyperthermia Against Human Prostate Cancer Cells

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21^WAF1/Cip, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.     

High-efficiency protein delivery into transfection-recalcitrant cell types

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.     

Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.     

Neurite Outgrowth of PC12 Cells by 4′-O-β-d-Glucopyranosyl-3′,4-Dimethoxychalcone from Brassica rapa L. ‘hidabeni’ was Enhanced by Pretreatment with p38MAPK Inhibitor

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.     

Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033.