Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Determination of eperisone in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric method was developed to determine eperisone hydrochloride, 4′-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride, in human plasma over the concentration range 0.2–40 ng/ml. Excellent sensitivity was achieved by selection of a favorable fragment ion, m/z 98, of eperisone and reduction of heat decomposition of eperisone by using a splitless injector and a shortened capillary column. The method described here allows the determination of plasma concentrations as low as 0.2 ng/ml, the concentration attained 6 h after a single oral administration of 50 mg. At eperisone hydrochloride concentrations higher than 0.5 ng/ml, the mean inter-day variation of accuracy of the assay was less than 12%.     

Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

NKT cells are responsible for the clearance of murine norovirus through the virus-specific secretory IgA pathway

Norovirus infection cause epidemic nonbacterial gastroenteritis in patients. The immune mechanisms responsible for the clearance of virus are not completely understood. We examined whether NKT cells are effective against norovirus infection using CD1d KO mice. The body weights of 4-weeks-old CD1d KO mice that were infected with murine norovirus-S7 (MNV-S7) were significantly lower than those of non-infected CD1d KO mice. On the other hand, the body weights of infected WT mice were comparable to those of non-infected WT mice. Correspondingly, CD1d KO mice had an almost 1000-fold higher MNV-S7 burden in the intestine after infection in comparison to WT mice. The mechanism responsible for the insufficient MNV-S7 clearance in CD1d KO mice was attributed to reduced IFN-γ production early during MNV-S7 infection. In addition, the markedly impaired IL-4 production in CD1d KO mice resulted in an impaired MNV-S7-specific secretory IgA production after MNV-S7 infection which is associated with mucosal immunity. Thus, the present results provide evidence that NKT cells play an essential role in MNV-S7 clearance.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Anti-Hiv-1 Activity of 2′,3′-Dideoxinucleoside Analogues : Structure-Activity Relationship

Abstract

Among the purine and pyrimidine 2′,3′-dideoxynucleosides, 2′,3′-didehydro-2′,3′-dideoxynucleosides, 3′-azido-2′,3′-dideoxynucleosides and 3′-fluoro-2′,3′-dideoxynucleosides, several congeners have been identified which achieve a potent and selective inhibition of HIV-1 replication in vitro.     

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.