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.     

Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates

Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.     

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.     

Arabidopsis carotenoid mutants demonstrate that lutein is not essential for photosynthesis in higher plants.

Lutein, a dihydroxy beta, epsilon-carotenoid, is the predominant carotenoid in photosynthetic plant tissue and plays a critical role in light-harvesting complex assembly and function. To further understand lutein synthesis and function, we isolated four lutein-deficient mutants of Arabidopsis that define two loci, lut1 and lut2 (for lutein deficient). These loci are required for lutein biosynthesis but not for the biosynthesis of beta, beta-carotenoids. The lut1 mutations are recessive, accumulate high levels of zeinoxanthin, which is the immediate precursor of lutein, and define lut1 as a disruption in epsilon ring hydroxylation. The lut2 mutations are semidominant, and their biochemical phenotype is consistent with a disruption of epsilon ring cyclization. The lut2 locus cosegregates with the recently isolated epsilon cyclase gene, thus, providing additional evidence that the lut2 alleles are mutations in the epsilon cyclase gene. It appears likely that the epsilon cyclase is a key step in regulating lutein levels and the ratio of lutein to beta,beta-carotenoids. Surprisingly, despite the absence of lutein, neither the lut1 nor lut2 mutation causes a visible deleterious phenotype or altered chlorophyll content, but both mutants have significantly higher levels of beta, beta-carotenoids. In particular, there is a stable increase in the xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) in both lut1 and lut2 mutants as well as an increase in zeinoxanthin in lut1 and beta-carotene in lut2. The accumulation of specific carotenoids is discussed as it pertains to the regulation of carotenoid biosynthesis and incorporation into the photosynthetic apparatus. Presumably, particular beta, beta-carotenoids are able to compensate functionally and structurally for lutein in the photosystems of Arabidopsis.     

The carotenoids of Flavobacterium strain R1560

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, -carotene (7,8,7,8-tetrahydro-, -carotene plus 7,8,11,12-tetrahydro-, -carotene), neurosporene, lycopene, -zeacarotene, -carotene, -carotene, -cryptoxanthin, rubixanthin, 3-hydroxy--zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of half-molecule reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.