Satoshi Fukuoka

A novel spore peptidoglycan hydrolase of Bacillus cereus: biochemical characterization and nucleotide sequence of the corresponding gene, sleL.

The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330-5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.

Mode of Action of a Germination-Specific Cortex-Lytic Enzyme, SleC, of Clostridium perfringens S40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

A history of microarrays in biomedicine

The fundamental strategy of the current postgenomic era or the era of functional genomics is to expand the scale of biologic research from studying single genes or proteins to studying all genes or proteins simultaneously using a systematic approach. As recently developed methods for obtaining genome-wide mRNA expression data, oligonucleotide and DNA microarrays are particularly powerful in the context of knowing the entire genome sequence and can provide a global view of changes in gene expression patterns in response to physiologic alterations or manipulation of transcriptional regulators. In biomedical research, such an approach will ultimately determine biologic behavior of both normal and diseased tissues, which may provide insights into disease mechanisms and identify novel markers and candidates for diagnostic, prognostic and therapeutic intervention. However, microarray technology is still in a continuous state of evolution and development, and it may take time to implement microarrays as a routine medical device. Many limitations exist and many challenges remain to be achieved to help inclusion of microarrays in clinical medicine. In this review, a brief history of microarrays in biomedical research is provided, including experimental overview, limitations, challenges and future developments.

Direct conversion of silver complexes to nanoscale hexagonal columns on a copper alloy for plasmonic applications

We introduced a novel method for the rapid synthesis of silver nanohexagonal thin columns from an aqueous mixture of sodium thiosulfate (Na2S2O3) and silver chloride (AgCl) simply added to a phosphor bronze substrate. The reaction is based on galvanic displacement and the products are potentially useful for plasmonic applications.

Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.

Molecular basis for endotoxin neutralization by amphipathic peptides derived from the α-helical cationic core-region of NK-lysin ☆

An analysis of the interaction of the NK-lysin derived peptide NK-2 and of analogs thereof with bacterial lipopolysaccharide (LPS, endotoxin) was performed to determine the most important biophysical parameters for an effective LPS neutralization. We used microcalorimetry, FTIR spectroscopy, Zeta potential measurements, and small-angle X-ray scattering to analyze the peptide:LPS binding enthalpy, the accessible LPS surface charge, the fluidity of the LPS hydrocarbon chains, their phase transition enthalpy change, the aggregate structure of LPS, and how these parameters are modulated by the peptides. We conclude that (i) a high peptide:LPS binding affinity, which is facilitated by electrostatic and hydrophobic interactions and which leads to a positive Zeta potential, (ii) the formation of peptide-enriched domains, which destabilize the lipid packing, demonstrated by a drastic decrease of phase transition enthalpy change of LPS, and (iii) the multilamellarization of the LPS aggregate structure are crucial for an effective endotoxin neutralization by cationic peptides.

Mechanism of Hbγ-35-induced an increase in the activation of the human immune system by endotoxins:

Endotoxins (LPS) are highly potent immune stimulatory molecules and are mainly known for triggering Gram-negative sepsis. However, besides their toxic effects, this stimulatory function may be advantageous, for example when used as an adjuvant during vaccination. Thus, there is always a narrow range between the useful wake-up of the immune system and its overwhelming reaction, which can lead to diseases like sepsis. This raises the question of which conformational properties are responsible for making the LPS aggregates more or less potent. As described previously, the size, type and form of LPS aggregates play a major role in their immune stimulatory activity. In this study we investigate the role of these parameters. On the one hand, we use a peptide (Pep19-2.5; Aspidasept) that causes a change of the LPS aggregate structure into a less toxic state; on the other hand, we use a potent immune stimulating peptide (Hbγ-35), leading to higher toxicity. We have found opposing effects on LPS aggregate conformations allowing a better understanding of the processes of immune stimulation.

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->

Effects of Edible Algae Polysaccharides on Allergic, Inflammatory, and Anti-Tumor Responses Through Toll-Like Receptor 4

Algae are eaten as healthy foods in Asian countries. We summarize our recent results on the immunoregulatory role of polysaccharide fractions from edible algae on immediate hypersensitivity, delayed-type hypersensitivity, and antitumor immune responses. They are divided into two types. One is to manipulate immune response through toll-like receptor 4 (TLR 4). The other uses different receptors to modify the immune response. Petalonia binghamiae polysaccharide fraction and Spirulina pacifica complex polysaccharide attenuate the delayed-type hypersensitivity and tumor growth by reducing the production of inflammatory cytokine, IL-17 through TLR4. This is suggested from the results that these polysaccharide fractions could suppress the delayed-type hypersensitivity and tumor growth in C3H/HeN but not in tolllike receptor 4 mutant, C3H/HeJ mice. Alginic acid, the polysaccharide from brown algae suppressed tumor growth in both C3H/HeN and C3H/HeJ mice and did not suppress delayed-type hypersensitivity response significantly, when administered intraperitoneally. We propose the potential usefulness of edible algae as the fine tuning reagents of the immune response. We also summarize the recent advancement in the area of regulation of immune responses in relation to these polysaccharides.