Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer

To target tumors overexpressing low-density lipoprotein receptors (LDLr), a pyropheophorbide cholesterol oleate conjugate was synthesized and successfully reconstituted into the low-density lipoprotein (LDL) lipid core. Laser scanning confocal microscopy studies demonstrated that this photosensitizer-reconstituted LDL can be internalized via LDLr by human hepatoblastoma G(2) (HepG(2)) tumor cells.     

Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis

Protective immunity against Mycobacterium tuberculosis infection requires the induction and maintenance of mycobacteria-specific, IFN-gamma-secreting CD4+ and CD8+ T lymphocytes. The development of Th1-like T cells is promoted by the early secretion and synergistic action of interleukin (IL)-12 and IL-18. This study compares the effects of plasmid-encoded IL-12 and IL-18 on the immunogenicity and protective efficacy of a DNA vaccine expressing the M. tuberculosis-secreted protein antigen 85B (DNA-85B). Co-immunization with either IL-12- or IL-18-expressing plasmids augmented the IFN-gamma-secreting T-cell response, and the maximum effect was observed with plasmids encoding both cytokines. Further the IL-12, but not the IL-18-expressing plasmid, significantly increased the protective efficacy of DNA-85B against pulmonary M. tuberculosis infection. Therefore co-administration of plasmid-encoded cytokines provides a potential method for optimizing the protective efficacy of DNA vaccination against tuberculosis.     

Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria

The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.     

Pyropheophorbide 2-deoxyglucosamide: a new photosensitizer targeting glucose transporters

To prepare near-infrared fluorescence imaging and photodynamic therapy agents targeted at glucose transporters, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG) was synthesized and evaluated in a 9L glioma rat model. Fluorescence imaging studies demonstrate that Pyro-2DG is selectively accumulated in the tumor. Upon its photoactivation, we demonstrate that this agent efficiently causes selective mitochondrial damage to the region of a tumor that was photoirradiated after administration of this agent, but does not affect tissues photoirradiated in the absence of the agent or tissues treated with the agent that are not photoirradiated. Preliminary confocal microscopy studies suggest that Pyro-2DG is delivered and trapped in tumor cells via the GLUT/hexokinase pathway and therefore is useful both as a tumor-targeted NIR fluorescence imaging probe and as a PDT agent for the destruction of cancer.     

Estimating divergence times in large phylogenetic trees

A new method, PATHd8, for estimating ultrametric trees from trees with edge (branch) lengths proportional to the number of substitutions is proposed. The method allows for an arbitrary number of reference nodes for time calibration, each defined either as absolute age, minimum age, or maximum age, and the tree need not be fully resolved. The method is based on estimating node ages by mean path lengths from the node to the leaves but correcting for deviations from a molecular clock suggested by reference nodes. As opposed to most existing methods allowing substitution rate variation, the new method smoothes substitution rates locally, rather than simultaneously over the whole tree, thus allowing for analysis of very large trees. The performance of PATHd8 is compared with other frequently used methods for estimating divergence times. In analyses of three separate data sets, PATHd8 gives similar divergence times to other methods, the largest difference being between crown group ages, where unconstrained nodes get younger ages when analyzed with PATHd8. Overall, chronograms obtained from other methods appear smoother, whereas PATHd8 preserves more of the heterogeneity seen in the original edge lengths. Divergence times are most evenly spread over the chronograms obtained from the Bayesian implementation and the clock-based Langley-Fitch method, and these two methods produce very similar ages for most nodes. Evaluations of PATHd8 using simulated data suggest that PATHd8 is slightly less precise compared with penalized likelihood, but it gives more sensible answers for extreme data sets. A clear advantage with PATHd8 is that it is more or less instantaneous even with trees having several thousand leaves, whereas other programs often run into problems when analyzing trees with hundreds of leaves. PATHd8 is implemented in freely available software.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.     

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.