Haruo Tanaka

Nanomycins, new antibiotics produced by a strain of Streptomyces. II. Structure and biosynthesis.

Evidence is put forward which describes the structure and stereochemistry of new antibiotics, nanaomycins A and B, as I and V, respectively. In order to study biosynthesis and to determine the position of the hydroxyl group in the naphthoquinone moiety, a feeding experiment with 1-13C-acetate was effectively carried out. Nanaomycins A and B were found to be synthesized from acetate via a polyketide by Streptomyces rosa var. notoensis.

Mechanism by which the lectin actinohivin blocks HIV infection of target cells

Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Manα(1–2)Man, Manα(1–2)Manα(1–2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (Kd = 3.4 × 10−8 M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the “cluster effect” of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.

Isolation and structure of nanaomycin D, an enantiomer of the antibiotic kalafungin

The absolute configuration of nanaomycin D, the fourth antibiotic isolated from Streptomyces rosa var. notoensis which is also obtained from nanaomycin A by air oxidation in methanol, has been determined; it has been found to be the enantiomer (1) of kalafungin (2).

Actinohivin, a Broadly Neutralizing Prokaryotic Lectin, Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

ABSTRACT The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (KD) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (α1,2-mannose)3 oligosaccharide but not by α1,3/α1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of α1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (∼5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4+ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.

Minimum of the order parameter fluctuations of seismicity before major earthquakes in Japan

It has been shown that some dynamic features hidden in the time series of complex systems can be uncovered if we analyze them in a time domain called natural time χ. The order parameter of seismicity introduced in this time domain is the variance of χ weighted for normalized energy of each earthquake. Here, we analyze the Japan seismic catalog in natural time from January 1, 1984 to March 11, 2011, the day of the M9 Tohoku earthquake, by considering a sliding natural time window of fixed length comprised of the number of events that would occur in a few months. We find that the fluctuations of the order parameter of seismicity exhibit distinct minima a few months before all of the shallow earthquakes of magnitude 7.6 or larger that occurred during this 27-y period in the Japanese area. Among the minima, the minimum before the M9 Tohoku earthquake was the deepest. It appears that there are two kinds of minima, namely precursory and nonprecursory, to large earthquakes.

HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity.

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AHs candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AHs anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AHs cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.

Verticilide, a new ryanodine-binding inhibitor, produced by Verticillium sp. FKI-1033.

A new ryanodine-binding inhibitor, verticilide, was isolated from the cultured broth of a fungus, Verticillium sp. FKI-1033. It is a 24-membered ring cyclic depsipeptide, its structure being elucidated as cyclo[(2R)-2-hydroxyheptanoyl-N-methyl- L-alanyl]4. Verticilide inhibited ryanodine binding to ryanodine receptors in the cockroach at an IC50 value of 4.2u2009μM, whereas inhibition against mouse ryanodine receptors was weak (IC50=53.9u2009μM).

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity.

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.

Phthoxazolin A, a specific inhibitor of cellulose biosynthesis from microbial origin. II. Isolation, physico-chemical properties, and structural elucidation.

Phthoxazolin A is a new inhibitor of cellulose biosynthesis produces by Streptomyces sp. OM-5714. The active compound was isolated, and the structure was elucidated by spectrometric analyses.

HIV-1 envelope trimer has similar binding characteristics for carbohydrate-binding agents as monomeric gp120

gp120 binds to AH by surface plasmon resonance (View Interaction: 1, 2)