Porphyrins in the perienteric fluid of Ascaris lumbricoides.

Cain G. D. and Bassow F. 1976. Porphyrins in the perienteric fluid of Ascaris lumbricoides. International Journal for Parasitology6: 79–82. Porphyrins in the perienteric fluid of adult female A. lumbricoides were esterified in methanolic H₂SO₄, extracted in chloroform, separated by thin-layer chromatography, and identified spectrophotometrically before and after conversion to their zinc and copper chelates. Protoporphyrin IX was the major component, comprising 95·4% of the total; the remaining 4·6 % was coproporphyrin III. Uroporphyrin was not detected; no porphyrins were recovered from other worm tissues. Fluid from worms with light and dark colored guts varied in protoporphyrin content from 0·58 to 4·08 nmoles/ml, respectively, but fluid from both groups contained similar molar ratios of protoporphyrin, coproporphyrin and heme.     

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites (Ile(116), Arg(175,) Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-d-cyclohexylalanine-cyclohexylalanine-d-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 (peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-d-cyclohexylalanine-Trp-Arg] (peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.     

In vivo intermembrane transfer of phospholipids in the photosynthetic bacterium Rhodopseudomonas sphaeroides.

The kinetics of accumulation of phospholipids into the intracytoplasmic membrane of Rhodopseudomonas sphaeroides have been examined. We have previously demonstrated that accumulation of phospholipids in the intracytoplasmic membrane is discontinuous with respect to the cell cycle. In this study we demonstrated a sevenfold increase in the rate of phospholipid incorporation into the intracytoplasmic membrane concurrent with the onset of cell division. Pulse-chase labeling studies revealed that the increase in the rate of phospholipid accumulation into the intracytoplasmic membrane results from the transfer of phospholipid from a site other than the intracytoplasmic membrane, and that the transfer of phospholipid, rather than synthesis of phospholipid, is most likely subject to cell cycle-specific regulation. The rates of synthesis of the individual phospholipid species (phosphatidylethanolamine, phosphatidyglycerol, and an unknown phospholipid) remained constant with respect to one another throughout the cell cycle. Similarly, each of these phospholipid species appeared to be transferred simultaneously to the intracytoplasmic membrane. We also present preliminary kinetic evidence which suggested that phosphatidylethanolamine may be converted to phosphatidycholine within the intracytoplasmic membrane.     

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.     

Abrupt Decline in Tuberculosis among Foreign-Born Persons in the United States

While the number of reported tuberculosis (TB) cases in the United States has declined over the past two decades, TB morbidity among foreign-born persons has remained persistently elevated. A recent unexpected decline in reported TB cases among foreign-born persons beginning in 2007 provided an opportunity to examine contributing factors and inform future TB control strategies. We investigated the relative influence of three factors on the decline: 1) changes in the size of the foreign-born population through immigration and emigration, 2) changes in distribution of country of origin among foreign-born persons, and 3) changes in the TB case rates among foreign-born subpopulations. Using data from the U.S. National Tuberculosis Surveillance System and the American Community Survey, we examined TB case counts, TB case rates, and population estimates, stratified by years since U.S. entry and country of origin. Regression modeling was used to assess statistically significant changes in trend. Among foreign-born recent entrants (<3 years since U.S. entry), we found a 39.5% decline (-1,013 cases) beginning in 2007 (P<0.05 compared to 2000–2007) and ending in 2011 (P<0.05 compared to 2011–2014). Among recent entrants from Mexico, 80.7% of the decline was attributable to a decrease in population, while the declines among recent entrants from the Philippines, India, Vietnam, and China were almost exclusively (95.5%–100%) the result of decreases in TB case rates. Among foreign-born non-recent entrants (≥3 years since U.S. entry), we found an 8.9% decline (-443 cases) that resulted entirely (100%) from a decrease in the TB case rate. Both recent and non-recent entrants contributed to the decline in TB cases; factors contributing to the decline among recent entrants varied by country of origin. Strategies that impact both recent and non-recent entrants (e.g., investment in overseas TB control) as well as those that focus on non-recent entrants (e.g., expanded targeted testing of high-risk subgroups among non-recent entrants) will be necessary to achieve further declines in TB morbidity among foreign-born persons.     

The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

Regulation of Listeria virulence: PrfA master and commander

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.