Akira Yano

Different frequencies of Streptococcus anginosus infection in oral cancer and esophageal cancer

Multiple cancers frequently occur in the upper aerodigestive tract. The high incidence rate of multiple carcinomas in this region is often explained in terms of involvement of the same underlying risk factors. It has been reported that the oral bacterium Streptococcus anginosus (S. anginosus) is associated with esophageal, gastric, and pharyngeal cancer tissues. In this study, a highly specific quantification method for S. anginosus DNA using real‐time PCR was established. We employed this assay to determine whether S. anginosus is also associated with oral cancer tissues. This precise quantification method revealed different degrees of infection with S. anginosus in esophageal cancer and oral cancer. We assayed 10 ng of genomic DNA from cancer tissues, and found that eight of 18 samples (44%) from the esophagus contained a detectable level (>10 fg) of S. anginosus DNA, whereas this was the case for only five of 38 samples (13%) of oral cancer. The quantity of S. anginosus DNA in the esophageal cancer tissues was significantly higher than in oral cancer. The maximum amount of S. anginosus DNA was approximately ten times higher in esophageal than in oral cancer tissues. In addition, none of the five different oral cancer sites (floor of the mouth, mandibular gingival, maxillary gingival, buccal mucosal, and tongue) showed significant signs of S. anginosus infection. On the other hand, most non‐cancerous tissues of the esophagus and tongue showed an undetectable level of S. anginosus. These results suggest that S. anginosus is associated with esophageal cancer, but is not closely related with oral cancer.

RGD motif enhances immunogenicity and adjuvanicity of peptide antigens following intranasal immunization

The use of peptides for various aspects of medical science has been a significant advance. Peptide-based vaccines are promising, but weak immunogenic potency is impeding the clinical application. We have remarkably enhanced the immunogenicity of peptide antigens by addition of motifs that bind to cell attachment proteins, such as arginine-glysine-aspartate (RGD), to the amino acid sequence. The modified peptides induced antigen-specific serum antibodies by intranasal immunization without adjuvants. RGD, an integrin-binding motif was the strongest, among several molecules tested in this experiment, giving an average of 10 times enhancement of antibody titers when incorporated into several peptide antigens. The peptides also acted as an efficient adjuvant following the intranasal immunization with protein antigens. Our data support the feasibility of developing peptide vaccines and peptide adjuvants for intranasal vaccination.

A chimeric laccase with hybrid properties of the parental Lentinula edodes laccases

We created a chimeric laccase from two different laccases, Lcc1 and Lcc4, from Lentinula edodes. Lcc1 is a secretory lignin-degrading enzyme produced in liquid cultures of L. edodes. Lcc4 is a tissue-accumulating-type enzyme, which is thought to be involved in melanin synthesis in fruiting body after harvesting. Lcc1 and Lcc4 differ in their Km values for some substrates, especially beta-(3,4-dihydroxyphenyl) alanine (L-DOPA) and catechol. The novel chimeric laccase, Lcc4/1, has properties that are a hybrid of those of Lcc1 and Lcc4. Lcc4/1 acts upon both Lcc1 and Lcc4 substrates and most of its Km values are lower than those of Lcc1 and Lcc4. Homology modeling indicates that the deduced shape of the substrate-binding pocket of the chimeric laccase is larger than that of Lcc1 and similar to that of Lcc4. The other biochemical properties, such as temperature and pH dependency, are intermediate between those of Lcc1 and Lcc4.

Effective induction of pblac1 laccase by copper ion in Polyporus brumalis ibrc05015

Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.

High cell-density expression system: a novel method for extracellular production of difficult-to-express proteins.

Yeasts extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.

Characterization of an extracellular laccase, PbLac1, purified from Polyporus brumalis

Polyporus brumalis (strain ibrc05015) secreted high amounts of laccases (Lacs) in liquid medium. With 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate, Lac activity was 7.72 U ml⁻¹ and this strain secreted a maximum 0.23 mg ml⁻¹ of total protein. The enzyme, PbLac1 was purified to homogeneity using hydrophobic and anion-exchange chromatography. The purified PbLac1 had a molecular mass of 63.4 kDa as determined by polyacrylamide-gel electrophoresis. PbLac1 oxidized a wide range of substrates such as 3,4-dihydroxy l-phenylalanine (l-DOPA) and catechol, but not tysorine. The activity of PbLac1 was increased by addition of 10.0 mM Cu(2+). PbLac1 could decolorize several industrial dyes, such as Remazol Brilliant Blue R known as model dyes of environmental xenobiotics. In addition, PbLac1 decolorized a wide range of substrates, such as the carcinogen, Poly R-478, in the presence of violuric acid as mediator. The E° value of PbLac1 was 0.80 V±0.01 versus normal hydrogen electrode, which is a very high redox potential compared to those of other basidiomycetous Lacs. These results suggest the potential utility of PbLac1 for industrial applications.

Transgenic plant-derived pharmaceuticals – the practical approach?

Production of biopharmaceuticals in transgenic plants would involve the creation of a new industry. Those transgenic plants, including staple food crops, could provide many benefits to people all over the world. However, the new industry might require a strict regulation system. It is probable that such a strict system would not be acceptable to Japan or to most developing countries. Many countries should use non-food crops for production of biopharmaceuticals and take on more simple systems. The new industry must develop strategies for promoting the benefits of transgenic plant-derived biopharmaceuticals on both the domestic and worldwide scales.

Identification and characterization of CcCTR1, a copper uptake transporter-like gene, in Coprinopsis cinerea.

Copper (Cu) is an essential element for the physiological function of organisms. In basidiomycetes, Cu is necessary for the production of phenol oxidase enzymes such as laccase and tyrosinase. We isolated and characterized two genes, CcCTR1 and -2, from the model basidiomycete Coprinopsis cinerea. CcCTR1 and -2 showed similarity to the Cu transporter CTR1 in Saccharomyces cerevisiae. Both CcCTRs had a MLxxM motif that is conserved in other CTR homologs. The addition of Cu to a liquid culture of C. cinerea decreased the mRNA accumulation of CcCTR1 and -2. Heterologous expression of CcCTR1 in S. cerevisiae increased Cu sensitivity, suggesting that CcCTR1 is a Cu uptake transporter. Together, these results suggest that CcCTR1 plays an important role in Cu accumulation in C. cinerea.

The Effect of Eating Sea Cucumber Jelly on Candida Load in the Oral Cavity of Elderly Individuals in a Nursing Home

We conducted a double-blind randomized controlled study of elderly individuals in a nursing home to investigate the effect of the consumption of jelly containing sea cucumber on their oral Candida load. The jelly contained a hydrolysate of the sea cucumber Stichopus japonicus, which contained triterpene glycosides called holotoxins. The holotoxins worked as a fungicide, and their minimum inhibitory concentrations for Candida albicans were 7 µg/mL. Eight individuals in the nursing home took the sea cucumber jelly for a week and their oral Candida were counted before and after the intervention. Nine individuals took a control jelly without S. japonicus. The sea cucumber jelly showed inhibitory effects on the oral Candida. Thus, daily consumption of the S. japonicus jelly has the potential to reduce the oral Candida load in the elderly in nursing homes.

The Peptide Vaccine Combined with Prior Immunization of a Conventional Diphtheria-Tetanus Toxoid Vaccine Induced Amyloid β Binding Antibodies on Cynomolgus Monkeys and Guinea Pigs

The reduction of brain amyloid beta (Aβ) peptides by anti-Aβ antibodies is one of the possible therapies for Alzheimers disease. We previously reported that the Aβ peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT) induced anti-Aβ antibodies, and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptide. Cynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination. Vaccination with a similar regimen was also performed on guinea pigs. The peptide vaccine induced anti-Aβ antibodies in cynomolgus monkeys and guinea pigs without chemical adjuvants, and excessive immune responses were not observed. Those antibodies could preferentially recognize Aβ 40, and Aβ 42 compared to Aβ fibrils. The levels of serum anti-Aβ antibodies and plasma Aβ peptides increased in both animals and decreased the brain Aβ 40 level of guinea pigs. The peptide vaccine could induce a similar binding profile of anti-Aβ antibodies in cynomolgus monkeys and guinea pigs. The peptide vaccination could be expected to reduce the brain Aβ peptides and their toxic effects via clearance of Aβ peptides by generated antibodies.