The Listeria monocytogenes DnaK chaperone is required for stress tolerance and efficient phagocytosis with macrophages

Listeria monocytogenes is a facultative intracellular pathogen which can escape bactericidal mechanisms and grow within macrophages. The intracellular environment of macrophages is one of the most stressful environments encountered by an invading bacterium during the course of infection. To study the role of the major stress protein, DnaK, of L. monocytogenes in survival under intracellular stress induced by macrophage-phagocytosis as well as under extracellular environmental stresses, we cloned, sequenced, and analyzed the dnaK locus from L. monocytogenes. Then we constructed an insertional mutation in the dnaK gene by homologous recombination and characterized it. Sequencing has revealed that the dnaK locus consists of four open reading frames in the order hrcA-grpE-dnaK-dnaJ. The mutant grows neither at temperatures above 39 degrees C nor under acidic conditions e.g. pH 3.0. Using the macrophage cell line JA-4, the ability of the dnaK mutant to grow intracellularly was examined. Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compared to that of intracellular wild type bacteria. However, following a 1-3 h latency period, the mutant multiplied in a similar fashion to the wild type within macrophage cells. A quantitative analysis of intracellular bacteria in macrophage cells by microscope and a binding assay of bacteria to the surface of macrophages by ELISA revealed that the lower number of viable dnaK mutant in macrophages after phagocytosis is due to the low efficiency of phagocytosis resulting from the reduced binding capacity of the dnaK mutant. These results demonstrate that DnaK of L. monocytogenes is essentially required for survival under high temperatures and acidic conditions. Though it does not largely contribute to the survival of L. monocytogenes in macrophage cells, it is essential for efficient phagocytosis. This is the first evidence that DnaK is required for the efficient phagocytosis of a facultative intracellular pathogen with macrophages.     

Essential structure of orexin 1 receptor antagonist YNT-707, part III: Role of the 14-hydroxy and the 3-methoxy groups in antagonistic activity toward the orexin 1 receptor in YNT-707 derivatives lacking the 4,5-epoxy ring

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX₁R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX₁R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX₁R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.     

4-deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-β-D-xylopyranosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-reactive triple bond-containing hydrophobic agents in the presence of Cu(2+) salt/ascorbic acid, a step known as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén conditions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of α:β-isomers of twelve derivatives were then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat various human diseases in which glycosaminoglycans participate.     

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12 ng within 45 min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation.     

Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10 mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1 min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.     

Protocol for the production of viable bimaternal mouse embryos

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.     

Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1-3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an ∼50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.