Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication

Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.     

DNA-dependent protein kinase (DNA-PK) inhibitors: structure-activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC₅₀ = 8 nM).     

Synthesis and antitumor activity of some indeno[1,2-b]quinoline-based bis carboxamides

A series of bis(11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamides) linked through the 6-carboxamides were prepared by coupling the requisite acid imidazolides with various diamines. Compounds with mono-cationic linker chains were more potent cytotoxins than the corresponding monomer in a panel of rodent and human cell lines, while those with the dicationic linker chains (CH2)2NR(CH2)2NR(CH2)2 and (CH2)2NR(CH2)3NR(CH2)2 showed extraordinarily high potencies (for example, IC50s of 0.18-1.4 nM against human Jurkat leukemia; up to 1000-fold more potent than the parent monomer). As seen previously in the monomeric series, small, lipophilic 4-substituents significantly increased potency in cell culture. The dimeric compounds were all slightly to significantly more potent in the mutant JL(A) and JL(D) cell lines that under-express topo II, suggesting that this enzyme is not their primary target. An 11-imino-linked dimer was much less active, and an asymmetric indeno[1,2-b]quinoline-6-carboxamide/naphthalimide dimer was less active than the comparable symmetric bis(indeno[1,2-b]quinoline-6-carboxamide). Selected analogues were active against sub-cutaneously implanted colon 38 tumors in mice, giving growth delays comparable to that of the clinical topo I inhibitor irinotecan at up to 10-fold lower doses. These compounds form an interesting new class of putative topo I inhibitors.     

Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Mixotrophy in ciliates

Mixotrophy is the occurrence of phagotrophy and phototrophy in the same organism. In ciliates the intracellular phototroph can be unicellular green algae (zoochlorellae), dinoflagellates (zooxanthellae), cryptomonads or sequestered chloroplasts from ingested algae. An intermediate mixotrophic mechanism is that where the phagotroph ingests algal cells, maintains them intact and functional in the cytoplasm for some time, but the algae are afterwards digested. This seems to occur in some species of Mesodinium. Ciliates with phototrophic endosymbionts have evolved independently in marine and freshwater habitats. The enslaved algal cells or chloroplasts provide host cells with organic matter. Mixotrophs flourish in oxygen-rich, but also in micro-aerobic waters and in the complete absence of oxygen. In the latter case, the aerobic host retains aerobic metabolism, sustained by the oxygen produced by the phototrophic endosymbionts or the sequestered chloroplasts. Mixotrophic ciliates can attain spectacular abundances in some habitats, and entirely dominate the ciliate community.     

Peripheral NK1.1 NKT cells are mature and functionally distinct from their thymic counterparts

One interesting aspect of NKT cell development is that although they are thymus dependent, the pivotal transition from NK1.1(-) to NK1.1(+) can often take place after immature NK1.1(-) NKT cells are exported to the periphery. NK1.1(-) NKT cells in general are regarded as immature precursors of NK1.1(+) NKT cells, meaning that peripheral NK1.1(-) NKT cells are regarded as a transient, semimature population of recent thymic emigrant NKT cells. In this study, we report the unexpected finding that most NK1.1(-) NKT cells in the periphery of naive mice are actually part of a stable, mature and functionally distinct NKT cell population. Using adult thymectomy, we show that the size of the peripheral NK1.1(-) NKT cell pool is maintained independently of thymic export and is not the result of NK1.1 down-regulation by mature cells. We also demonstrate that most peripheral NK1.1(-) NKT cells are functionally distinct from their immature thymic counterparts, and from NK1.1(+) NKT cells in the periphery. We conclude that the vast majority of peripheral NK1.1(-) NKT cells are part of a previously unrecognized, mature NKT cell subset.