Tadazumi Komiyama

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism.

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-β-d-glucopyranoside and 4′-O-methyl delphinidin 3-O-β-d-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-β-d-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the - and ONOO− -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin ≈ cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO− was: delphinidin > cyanidin ≈ petunidin > malvidin ≈ (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4′-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO− -scavenging activities among the anthocyanins in the mixture.

Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status.

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Parkinsons, and Alzheimers diseases. Moreover, emotional stress effects in the central nervous system play a vital role in homeostasis. The protective effect of anthocyanins on the cerebral oxidative stress was studied using the whiskers cut model. In mice, such treatment causes psychological or emotional distress leading to oxidative stress in tissues. To investigate the in vivo antioxidant activity of anthocyanins, an extract of Vaccinium myrtillis L., an anthocyanin mixture, was orally administered (100 mg/kg of body weight.) to mice for 7 days, and then psychological stress was assessed by cutting off their whiskers. Whisker removal increased both protein carbonyl formation and lipid peroxidation in the brain, heart, kidney, and liver. Further, the levels of oxidative markers showed regional differences in the brain. Concomitantly, dopamine neurotransmitter levels were altered in both the midbrain and the brain cortex. Orally administered anthocyanins were also active in the brain, suppressing stress-induced cerebral oxidative stress and dopamine abnormalities in distressed mice. These effects of anthocyanin treatment suggest their possible usefulness for the treatment of cerebral disorders related to oxidative stress.

Inhibition of Fungal β-1,3-Glucan Synthase and Cell Growth by HM-1 Killer Toxin Single-Chain Anti-Idiotypic Antibodies

ABSTRACT Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis. scFv antibodies were fungicidal at concentrations of 1.56 to 12.5 μg/ml in vitro against four Candida species. The scFv antibodies exerted a strong candidacidal activity in vitro, with 50% inhibitory concentration (IC50) values ranging from 7.3 × 10−8 to 16.0 × 10−8 M, and were neutralized by adsorption with nMAb-KT. Furthermore, all scFv antibodies effectively inhibited fungal β-1,3-glucan synthase activity in vitro, with IC50 values ranging from 2.0 × 10−8 to 22.7 × 10−8 M, values which almost coincide with the values that are inhibitory to the growth of fungal cells. Binding assays showed that the scFv antibodies specifically bind to nMAb-KT, and this binding pattern was confirmed by surface plasmon resonance analysis. The binding ability was further demonstrated by the competition observed between scFv antibodies and HM-1 to bind nMAb-KT. To the best of our knowledge, this is the first study to show that an antifungal anti-idiotypic antibody, in the form of recombinant scFv, potentially inhibits β-1,3-glucan synthase activity.

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody.

BackgroundPhage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies.ResultsTo produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal antibodies (nmAb-KT), the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50) showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48%) of these clones (scFv K1) were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (Kd value = 4.62 × 10-8 M) had strong reactivity toward nmAb-KT, like HM-1 (Kd value = 6.74 × 10-9 M) as judged by SPR analysis.ConclusionThe scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.

Synthesis, anti-fungal and 1,3-β-D-glucan synthase inhibitory activities of caffeic and quinic acid derivatives

New derivatives of caffeic acid and quinic acid were synthesized and their anti-fungal and inhibitory activities on fungal 1,3-β-glucan synthase were determined in comparison with those of the corresponding chlorogenic acid derivatives. All the chlorogenic, quinic and caffeic acid derivatives that were coupled with an H(2)N-orn-4-(octyloxy) aniline group (1, 1b and 1c) displayed potent activities in both anti-fungal and inhibition of 1,3-glucan synthase assays. Compounds 1 and 1c inhibited the fungal membrane enzyme with the potency comparable to that of a known 1,3-β-D-glucan synthase inhibitor, aculeacin A. The results revealed that the anti-fungal activity of the chlorogenic acid derivative with a free amino group was at least partly due to inhibition of the fungal 1,3-β-glucan synthase. These results suggest that further investigation on caffeic acid derivatives may lead to the discovery of novel anti-fungal agents with drug-like properties.

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution.

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1+HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC(50) values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40microM and 2.20microM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K(d) value of 3.09x10(-11)M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.

Inhibition of β-1,3-Glucan Synthase and Cell Growth of Cryptococcus species by Recombinant Single-chain Anti-idiotypic Antibodies

Recombinant single-chain fragment variable (scFv) anti-idiotypic antibodies were produced to represent the internal image of a HM-1 killer toxin, which is characterized by a wide spectrum of anti-fungal activity through inhibiting β-1,3-glucan synthase (GS). We examined if scFv antibodies are active against Cryptococcus species, a human pathogen of increasing medical importance. The anti-cryptococcal activity of scFv antibodies and HM-1 were assessed by MIC analysis for C. neoformans IFM 40215 and C. albidus NBRC 0612 cells. The scFv antibodies had strong anti-cryptococcal activity in vitro with IC50 at 1.07×10−7 to 2.60×10−7 M for C. neoformans and C. albidus. Furthermore, the scFv antibodies potentially inhibited GS of C. neoformans with IC50 at 1.27×10−7 to 2.27×10−7 M. Both the anti-fungal and anti-GS activities of the scFv antibodies were markedly neutralized by the monoclonal antibody that neutralizes HM-1 killer toxin.

Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.

Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

The high-osmolarity glycerol- and cell wall integrity-MAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM-1.

Fps1p is an aquaglyceroporin important for turgor regulation of Saccharomyces cerevisiae. Previously we reported the involvement of Fps1p in the yeast‐killing action of killer toxin HM‐1. The fps1 cells showed a high HM‐1‐resistant phenotype in hypotonic medium and an HM‐1‐susceptible phenotype in hypertonic medium. This osmotic dependency in HM‐1 susceptibility was similar to those observed in Congo red, but different from those observed in other cell wall‐disturbing agents. These results indicate that HM‐1 exerts fungicidal activity mainly by binding and inserting into the yeast cell wall structure, rather than by inhibiting 1,3‐β‐glucan synthase. We next determined HM‐1‐susceptibility and diphospho‐MAP kinase inductions in S. cerevisiae. In the wild‐type cell, expressions of diphospho‐Hog1p and ‐Slt2p, and mRNA transcription of CWP1 and HOR2, were induced within 1 h after an addition of HM‐1. ssk1 and pbs2 cells, but not sho1 and hkr1 cells, showed HM‐1‐sensitive phenotypes and lacked inductions of phospho‐Hog1p in response to HM‐1. mid2, rom2 and bck1 cells showed HM‐1‐sensitive phenotypes and decreased inductions of phospho‐Slt2p in response to HM‐1. From these results, we postulated that the Sln1–Ypd1–Ssk1 branch of the high‐osmolality glycerol (HOG) pathway and plasma membrane sensors of the cell wall integrity (CWI) pathway detect cell wall stresses caused by HM‐1. We further suggested that activations of both HOG and CWI pathways have an important role in the adaptive response to HM‐1 toxicity. Copyright

Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.

Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.