Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.     

A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC₅₀ = 19 nM) in VLA-4 inhibitory activity compared to 1 (IC₅₀ = 1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC₅₀ = 2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL = 3.3 ml/min/kg, F = 51%) in rats.     

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.     

Ganodermasides C and D, two new anti-aging ergosterols from spores of the medicinal mushroom Ganoderma lucidum

Two new anti-aging compounds, ganodermasides C and D, were isolated and their structures elucidated. They are novel ergosterols possessing a 4,6,8(14),22-tetraene-3-one unit with unique hydroxylation at C-9. Both of them significantly extended the replicative lifespan of the K6001 yeast strain. Ganodermasides C and D regulated the expression of the gene for UTH1 to prolong the replicative lifespan of yeast.     

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K_i = 0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K_i value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K_i values compared to the wild-type enzyme, while the remaining mutations had little or no effects.     

Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. 1: Synthesis of triplet drugs with morphinan skeletons

We synthesized symmetrical and nonsymmetrical triplet drugs with 1,3,5-trioxazatriquinane skeletons. The isolation of key intermediates, oxazoline dimers, made it possible to effectively produce nonsymmetrical triplets. Among the synthesized triplets, KNT-93, composed of three identical opioid μ receptor agonists, showed dose-dependent antinociception via the μ receptor. The effect was 56-fold more potent than that of morphine, a representative μ agonist. The profound analgesic effect induced by KNT-93 might result from simultaneous occupation of three μ opioid receptors.     

Plasma free amino acid profiling of five types of cancer patients and its application for early detection

Background

Recently, rapid advances have been made in metabolomics-based, easy-to-use early cancer detection methods using blood samples. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. Furthermore, PFAA profiles are known to be influenced by specific diseases, including cancers. Therefore, the purpose of the present study was to determine the characteristics of the PFAA profiles in cancer patients and the possibility of using this information for early detection.

Methods and Findings

Plasma samples were collected from approximately 200 patients from multiple institutes, each diagnosed with one of the following five types of cancer: lung, gastric, colorectal, breast, or prostate cancer. Patients were compared to gender- and age- matched controls also used in this study. The PFAA levels were measured using high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS). Univariate analysis revealed significant differences in the PFAA profiles between the controls and the patients with any of the five types of cancer listed above, even those with asymptomatic early-stage disease. Furthermore, multivariate analysis clearly discriminated the cancer patients from the controls in terms of the area under the receiver-operator characteristics curve (AUC of ROC >0.75 for each cancer), regardless of cancer stage. Because this study was designed as case-control study, further investigations, including model construction and validation using cohorts with larger sample sizes, are necessary to determine the usefulness of PFAA profiling.

Conclusions

These findings suggest that PFAA profiling has great potential for improving cancer screening and diagnosis and understanding disease pathogenesis. PFAA profiles can also be used to determine various disease diagnoses from a single blood sample, which involves a relatively simple plasma assay and imposes a lower physical burden on subjects when compared to existing screening methods.     

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.     

Structural insights into differences in drug-binding selectivity between two forms of human alpha1-acid glycoprotein genetic variants, the A and F1*S forms

Human α(1)-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ.