Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease

BackgroundVisceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL.Methodology/Principal findingsSampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis.Conclusions/SignificanceSeroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.     

Antitumor agents. 258. Syntheses and evaluation of dietary antioxidant--taxoid conjugates as novel cytotoxic agents

Various dietary antioxidants, including vitamins, flavonoids, curcumin, and a coumarin, were conjugated with paclitaxel (1) through an ester linkage. The newly synthesized compounds were evaluated for cytotoxic activity against several human tumor cell lines as well as the corresponding normal cell lines. Interestingly, most tested conjugates selectively inhibited the growth of 1A9 (ovarian) and KB (nasopharyngeal) tumor cells without activity against other cell lines. Particularly, conjugates 16 and 20 were highly active against 1A9 (ED(50) value of 0.005 microg/mL) as well as KB (ED(50) values of 0.005 and 0.14 microg/mL, respectively) cells. Compound 22b, the glycinate ester salt of vitamin E conjugated with 1, appears to be a promising lead for further development as a clinical trial candidate as it exhibited strong inhibitory activity against Panc-1 (pancreatic cancer) with less effect on the related E6E7 (normal) cell line.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Amino acid changes in hemagglutinin contribute to the replication of oseltamivir-resistant H1N1 influenza viruses

Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008.     

Biochemical characterization of a N-terminal fragment (p5) cleaved from fibroblast growth factor-binding protein (FGF-BP) in bovine milk in vitro

By means of successive gel filtration on a Superdex 30 pg column and Mono S column chromatography, a 5-kDa polypeptide (p5) was highly purified from the low molecular weight (LMW) fraction separated from the partially purified lactoferrin (bLF) fraction of bovine milk, and biochemically characterized as a phosphate acceptor for two protein kinases [cAMP-dependent protein kinase (PKA) and casein kinase 1delta (CK1delta)] in vitro. Purified p5 was identified as a fragment (N-terminal positions 24-51, 28 amino acid residues) cleaved from fibroblast growth factor-binding protein (FGF-BP, p37). Both purified p5 and synthetic p5 (sp5) were effectively phosphorylated by PKA, and also phosphorylated by CK1delta in the presence of two sulfated lipids [sulfatide or cholesterol-3-sulfate (CH-3S), SCS] in vitro. A novel phosphorylation site (RNRRGS) for CK1delta and a potent SCS-binding site (RNRR) on p5 were identified. The PKA-mediated phosphorylation of p5 was highly stimulated when incubated with either acidic FGF (aFGF) or bLF in vitro, but this phosphorylation was more sensitive to SCS than H-89 (a specific PKA inhibitor). Immunoprecipitate experiments revealed p5, but not the phosphorylated p5, to be directly bound to aFGF in vitro. These results show that (i) p5 has a high binding affinity with aFGF as well as bLF; (ii) the binding of SCS to p5 results in the selective inhibition of its phosphorylation by PKA; and (iii) SCS functions as an effective stimulator for the phosphorylation of p5 by CK1delta in vitro. In addition, p5 may play an important physiological role as a trafficking factor for the physiological interaction between aFGF group including endothelial cell growth factors and their binding proteins in vivo.     

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.     

Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris

Two novel tachykinins (OctTK-I: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Ile-Gly-Leu-Met-NH(2) and OctTK-II: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Val-Gly-Leu-Met-NH(2)) were isolated from the posterior salivary gland of the octopus (Octopus vulgaris) using a contraction assay of the carp rectum. These peptides had in common the pentapeptide sequence -Phe-X-Gly-Leu-Met-NH(2) at the C-terminal and induced immediate contractions on the carp rectum and the guinea-pig ileum. cDNAs encoding their precursor proteins were cloned. The OctTK gene was expressed in the posterior salivary gland and the expression was localized in mucus-secreting cells of the gland. The results suggested that OctTKs might be secreted as a venomous substance acting on vertebrates such as fishes, which are the prey or natural enemies of the octopus.