Marine-derived microbes and molecules for drug discovery.

Increasing attention has been paid to marine-derived biomolecules as sources of therapeutics for autoimmune diseases. Nagasaki Prefecture has many islands and is surrounded by seas, straits, gulfs, bays, and coves, giving it the second longest coastline in Japan after Hokkaido. We have collected more than 20,000 marine microbes and have been preparing an original marine microbial extract library, which contains small and mid-size biomolecules that may penetrate cell membranes and interfere with the intracellular protein-protein interaction involved in the development of autoinflammatory diseases such as familial Mediterranean fever. In addition, we have been developing an indoor shark farming system to prepare shark nanobodies that could be developed as potential therapeutic agents for autoimmune diseases. Sharks produce heavy-chain antibodies, called immunoglobulin new antigen receptors (IgNARs), consisting of one variable domain (VNAR) and five constant domains (CNAR); of these, VNAR can recognize a variety of foreign antigens. A VNAR single domain fragment, called a nanobody, can be expressed in Escherichia coli and has the properties of an ideal therapeutic candidate for autoimmune diseases. Shark nanobodies contain complementarity-determining regions that are formed through the somatic rearrangement of variable, diversity, and joining segments, with the segment end trimming and the N- and P-additions, as found in the variable domains of mammalian antibodies. The affinity and diversity of shark nanobodies are thus expected to be comparable to those of mammalian antibodies. In addition, shark nanobodies are physically robust and can be prepared inexpensively; as such, they may lead to the development of highly specific, stable, effective, and inexpensive biotherapeutics in the future. In this review, we first summarize the history of the development of conventional small molecule drugs and monoclonal antibody therapeutics for autoimmune diseases, and then introduce our drug discovery system at Nagasaki University, including the preparation of an original marine microbial extract library and the development of shark nanobodies.

Classification of atrophic mucosal patterns on Blue LASER Imaging for endoscopic diagnosis of Helicobacter pylori-related gastritis: A retrospective, observational study.

BACKGROUND: Atrophic gastritis can be classified according to characteristic mucosal patterns observed by Blue LASER Imaging (BLI) in a medium-range to distant view. AIMS: To facilitate the endoscopic diagnosis of Helicobacter pylori (HP)-related gastritis, we investigated whether atrophic mucosal patterns correlated with HP infection based on the image interpretations of three endoscopists blinded to clinical features. METHODS: This study included 441 patients diagnosed as having atrophic gastritis by upper gastrointestinal endoscopy at Nishikawa Gastrointestinal Clinic between April 1, 2015 and March 31, 2016. The presence/absence of HP infection was not taken into consideration. Endoscopy was performed using a Fujifilm EG-L580NW scope. Atrophic mucosal patterns observed by BLI were classified into Spotty, Cracked and Mottled. Image interpretation results were that 89, 122 and 228 patients had the Spotty, Cracked and Mottled patterns, respectively, and 2 patients an undetermined pattern. Further analyses were performed on 439 patients, excluding the 2 with undetermined patterns. RESULTS: The numbers of patients testing negative/positive for HP infection in the Spotty, Cracked and Mottled pattern groups were 12/77, 105/17, and 138/90, respectively. The specificity, positive predictive value and positive likelihood ratio for endoscopic diagnosis with positive HP infection based on the Spotty pattern were 95.3%, 86.5% and 8.9, respectively. In all patients with the Spotty pattern before HP eradication, the Cracked pattern was observed on subsequent post-eradication endoscopy. CONCLUSIONS: The Spotty pattern may represent the presence of HP infection, the Cracked pattern, a post-inflammatory change as seen after HP eradication, and the Mottled pattern, intestinal metaplasia.

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation.

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.

Estrogenic activity removal of ethynylestradiol by nitrifying activated sludge and microorganisms involved in its degradation.

A solid-phase extraction of ethynylestradiol (EE2) and its degradation intermediates and products by nitrifying activated sludge (NAS) was performed to measure estrogenic activity during the degradation by NAS. The yeast two-hybrid assay of the extract showed that NAS not only degraded EE2, but also removed the estrogenic activity originating from EE2, its degradation intermediates and products. This means that estrogenic activity decreases when EE2 concentrations are decreased by NAS. Although gas chromatography/mass spectrometer (GC/MS) analyses suggested the existence of nitrated EE2 as a degradation intermediate, its estrogenic activity was unmeasurable due to the lack of an authentic sample. However, on the basis of the dose-response relationship between EE2 concentration and estrogenic activity in the yeast two-hybrid assay, it was surmised that the estrogenic activity in the culture medium of NAS may have been produced mostly by the remaining EE2 and not by its degradation intermediates and products. The partial nucleotide sequence analyses of microorganisms in the consortiums acclimatized for EE2 and natural estrogens E2 and E1 enabled the identification of candidates that may be responsible for estrogen degradation by NAS.

Apoptosis related to steroid sulfatase at the cone-shaped projection in nephron morphogenesis.

Steroid sulfatase (STS) is a microsomal enzyme catalysing the hydrolysis of steroid 3beta-sulfates, including estrogen sulfate, at organ target sites. Steroid sulfatase activity, as well as the activity of various estrogens, has been reported to influence the development of organs. In the kidney, progenitor cells of nephrons develop from mesenchymal cells and finally attach to the ureteric duct at the region termed the cone-shaped projection. However, the mechanisms of this process have not been fully investigated. In the present study, STS-related apoptosis occurring between the renal vesicle and ureteric duct was examined using immunohistochemical techniques. Immunoreactivity to STS was demonstrated at the cortical metanephric mesenchyme surrounding new vessels and was pronounced at the junction between the renal vesicle and ureteric duct (cone-shaped junction). Steroid sulfatase-transfected LLC-RK1 cells (rat proximal cell line) were also examined in vitro. Apoptosis occurred when an estrogen precursor (ELS) was added at concentrations between 10(-4) and 10(-2) mol/L. These results indicate that STS is synthesized in the progenitor cells of proximal tubules. Moreover, STS may be related to apoptosis occurring at the junction between the renal vesicle and ureteric duct in a manner proportional to the availability of STS precursors.

T-cell receptor antagonist modifies cytokine secretion profile of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells.

A T-cell receptor (TCR) antagonist is an analog of a peptide ligand for TCR that inhibits T-cell responses to the original peptide. We investigated the effects of a TCR antagonist on cytokine secretion of naive CD4+ T cells and their differentiation into type-1 and type-2 helper T cells (Th1 and Th2) induced by stimulation with varying doses of an antigenic peptide. In the presence of a TCR antagonist peptide, proliferation of naive CD4+ T cells and antigen dose-dependent secretion of interferon-gamma, a typical Th1-type cytokine, by these cells was down-regulated. With respect to the secretion of interleukin-4 (IL-4), a typical Th2-type cytokine, the TCR antagonist raised the concentration of the antigenic peptide required to elicit maximal IL-4 production and, surprisingly, significantly increased the maximum level of IL-4 secretion. Similar effects induced by the TCR antagonist were observed on the Th1/Th2 differentiation of naive CD4+ T cells. These results clearly indicate that, for naive CD4+ T cells, a TCR antagonist has the potential to change the balance of Th1/Th2 cytokine secretion and even enhance Th2 responses.

Characterization of sulfur exchange reaction between polysulfides and elemental sulfur using a 35S radioisotope tracer method.

A sulfur exchange reaction between di-tert-butylpolysulfides and elemental sulfur was examined using a 35S tracer method and the reaction mechanism was discussed.

In vitro characterization of Mato's FGP cells isolated from rat cerebrum.

Mato's FGP cells surrounding cerebral arterioles play a significant role in the maintenance of a homeostatic microenvironment in the brain. In this study, the perivascular cells were isolated from rat cerebral microvessels and cultured in vitro to characterize their phenotype. Autofluorescence of the intracellular granules in cultured cells and the uptake of HRP and DiI-Ac-LDL by these cells were observed. The cells reacted positively to an anti-scavenger receptor A antibody. Positive immunoreactions of cultured cells to ED1 and ED2 antibodies were observed, whereas they were weak or negative to ED3 and OX42 antibodies. Acid phosphatase activity was detected in the granules of cultured cells. In conclusion, the cells cultivated under the present conditions revealed very similar characteristics to Mato's FGP cells in situ and therefore are useful for studies on FGP cells.