Faculty Bibliography

The increasing recognition that symbioses have greatly altered evolution through genome fusions is creating a need for algorithms that can reliably detect and reconstruct fusions. Here, we generalize the bootstrappers gambit algorithm (a quartet method) in order to permit it to analyze both bifurcations and fusions under a single mathematical model, and thereby detect past genomic branchings and endosymbioses. This new method, 3-dimensional parsimony, can be applied to aligned sequences, such as gene, indel, or other genomic presence/absence sequences. It also provides a statistical measure of support for each possible graph. The usefulness of this method is demonstrated by applying it to the ring of life.

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and alpha(2)-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3-6 muM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from alpha(2)-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65-80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100-500 microM) inhibited copper uptake from albumin by 20-30% in both cell types and that from alpha(2)-macroglobulin by 0-30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. alpha(2)-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified.

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR(+) cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.

Gag-CD8+ T cell responses are associated with immune control of HIV infection. Since during HIV infection Nef impairs T cell responses, we evaluated whether deletion of nef from a non-infectious HIV DNA vaccine (Delta4 Nef+), creating Delta5 Nef(-), would affect its immunogenicity. When compared with Delta4, mice injected with Delta5 developed significantly lower CD8+ T cell responses to Gag, but no significant change in the responses to Env was observed. In vitro, deletion of Nef abrogated the induced cell death, production of virus-like particles and release of Gag from transfected cells. Thus, the effect of Nef in causing extrusion of Gag might adjuvant the CD8+ T cell responses to Gag in DNA vaccine.

In this study, the complete genomic sequence was determined for three hepatitis C virus variants (VT21, TV241 and TV249) of genotype 6 that do not classify within the established subtypes. All three genomes were isolated from patients in Vietnam and sequenced using 100 microl of serum. They showed 91.4-93.6% nucleotide similarities to each other but only 71.7-79.4% similarities to 17 reference sequences representing subtypes 6a-6q and to isolates km41 and gz52557. VT21, TV241 and TV249 displayed genome lengths of 9407, 9460 and 9445 nt, respectively. All three isolates contained a single open reading frame of 9051 nt while the 5'UTRs and 3'UTRs were 324-338 nt and 32-71 nt, respectively. They shared common sizes with QC227/6o and QC216/6p isolates in all ten protein regions. Phylogenetic analyses demonstrated that VT21, TV241 and TV249 clustered independently and were assigned subtype 6t, following the recent designations of 6r and 6s. Analysis of partial genomic sequences available for genotype 6 variants revealed five additional subtype 6t isolates, all originating from Vietnam. This analysis revealed two additional groups of isolates, and at least seven novel variants analogous to km41 and gz52557 that group independently and do not classify within the subtypes 6a-6t. This suggests the existence of at least 11 additional subtypes for genotype 6. In addition, the existence of isolates showing genetic distances greater than those within subtypes, but lesser than those between subtypes, raises interesting questions regarding the classification of HCV.

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.

Pancreatic carboxyl ester lipase (CEL) is in the plasma of many mammals, including humans and rats, but not mice. In vitro, CEL hydrolyzes cholesterol esters of apolipoprotein B-containing lipoproteins (apo B-Lp). To study the effect of CEL on metabolism of apo B-Lp and atherosclerosis in vivo, apo E-knockout (EKO) mice, which have high plasma levels of apo B-Lp and are prone to atherosclerosis, were made to secrete CEL into plasma by introducing a transgene containing a liver-specific promoter and rat CEL complementary DNA. Plasma CEL activity in EKO-CEL mice was comparable with that found in rats. Evidence of modification of apo B-Lp by plasma CEL in vivo was an increase in the free cholesterol to cholesterol ester ratio of apo B-Lp from mice on chow or a Western-type diet. In addition, plasma total cholesterol levels were elevated in EKO-CEL mice, with the elevation found exclusively in the apo B-Lp fraction. Associated with the increase in steady-state apo B-Lp levels was an increase in the plasma half-life of very low-density lipoproteins (VLDL) in EKO-CEL mice, measured by the clearance rate of injected VLDL. Interestingly, despite the increase of apo B-Lp, the atherosclerotic lesion did not differ between EKO and EKO-CEL mice on a Western-type diet. In summary, our results demonstrate that plasma CEL modulates apo B-Lp metabolism in vivo, resulting in reduced VLDL clearance and elevated plasma cholesterol levels.

Cardiovascular disease is one of the prime causes of mortality throughout the world and there is a need for targeted and effective contrast agents to allow noninvasive imaging of the cholesterol-rich atherosclerotic plaques in arteries. A new, fully synthetic, high-density lipoprotein (HDL)-mimicking MRI contrast agent is developed, which enhances macrophage-rich areas of plaque in a mouse model of atherosclerosis by 94%. Confirmation of the targeting of this nanoparticulate agent is achieved using confocal microscopy by tracking a fluorescent lipid incorporated into the nanoparticle.

Because the levels of secreted apolipoprotein B (apoB) directly correlate with circulating serum cholesterol levels, there is a pressing need to define how the biosynthesis of this protein is regulated. Most commonly, the concentration of a secreted, circulating protein corresponds to transcriptionally and/or translationally regulated events. By contrast, circulating apoB levels are controlled by degradative pathways in the cell that select the protein for disposal. This article summarizes recent findings on two apoB disposal pathways, endoplasmic reticulum (ER)-associated degradation and autophagy, and describes a role for post-ER degradation in the increased circulating lipid levels in insulin-resistant diabetics.