Microbial Species Involved in Production of 1,2-sn-Diacylglycerol and Effects of Phosphatidylcholine on Human Fecal Microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.     

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.     

Dynamics of putative raft-associated proteins at the cell surface

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.     

Alkyl lysophosphatidic acid and fluoromethylene phosphonate analogs as metabolically-stabilized agonists for LPA receptors

We describe an efficient method for the synthesis of alkyl lysophosphatidic acid (LPA) analogs as well as alkyl LPA mono- and difluoromethylene phosphonate analogs. Each alkyl LPA analog was evaluated for subtype-specific LPA receptor agonist activity using a cell migration assay for LPA(1) activation in cancer cells and an intracellular calcium mobilization assay for LPA(2) and LPA(3) activation. Alkyl LPAs induced pronounced cell migration activity with equivalent or higher potency than sn-1-oleoyl LPA, while the alkyl LPA fluoromethylene phosphonates proved to be less potent agonists in this assay. However, each alkyl LPA analog activated Ca(2+) release by activation of LPA(2) and LPA(3) receptors. Interestingly, the absolute configuration of the sn-2 hydroxyl group of the alkyl LPA analogs was not recognized by any of the three LPA receptors. The use of alkyl LPA analogs further expands the scope of structure-activity studies, which will better define LPA-LPA receptor interactions.     

Didehydrofarnesyl diphosphate: an intrinsically fluorescent inhibitor of protein farnesyltransferase

Didehydrofarnesyl diphosphate (delta delta FPP), a fluorescent pentaene analogue of farnesyl diphosphate (FPP), was synthesized using stereoselective Wittig reactions. Although delta delta FPP was not an alternative substrate for yeast protein farnesyltransferase (FTase), the fluorescent analogue was a potent competitive inhibitor with a K(i) value of 8.8 microM (K (m) (FPP) = 27 microM).     

A comparison of the 1998 and 2002 coral bleaching events on the Great Barrier Reef: spatial correlation,patterns, and predictions

Detailed mapping of coral bleaching events provides an opportunity to examine spatial patterns in bleaching over scales of 10 s to 1,000 s of km and the spatial correlation between sea surface temperature (SST) and bleaching. We present data for two large-scale (2,000 km) bleaching events on the Great Barrier Reef (GBR): one from 1998 and another from 2002, both mapped by aerial survey methods. We examined a wide range of satellite-derived SST variables to determine which one best correlated with the observed bleaching patterns. We found that the maximum SST occurring over any 3-day period (max3d) during the bleaching season predicted bleaching better than anomaly-based SST variables and that short averaging periods (3–6 days) predicted bleaching better than longer averaging periods. Short periods of high temperature are therefore highly stressful to corals and result in highly predictable bleaching patterns. Max3d SST predicted the presence/absence of bleaching with an accuracy of 73.2%. Large-scale (GBR-wide) spatial patterns of bleaching were similar between 1998 and 2002 with more inshore reefs bleached compared to offshore reefs. Spatial change in patterns of bleaching occurred at scales of ~10 s km, indicating that reefs bleach (or not) in spatial clusters, possibly due to local weather patterns, oceanographic conditions, or both. Approximately 42% of reefs bleached to some extent in 1998 with ~18% strongly bleached, while in 2002, ~54% of reefs bleached to some extent with ~18% strongly bleached. These statistics and the fact that nearly twice as many offshore reefs bleached in 2002 compared to 1998 (41 vs. 21%, respectively) makes the 2002 event the worst bleaching event on record for the GBR. Modeling of the relationship between bleaching and max3d SST indicates that a 1 °C increase would increase the bleaching occurrence of reefs from 50% (approximate occurrence in 1998 and 2002) to 82%, while a 2 °C increase would increase the occurrence to 97% and a 3 °C increase to 100%. These results suggest that coral reefs are profoundly sensitive to even modest increases in temperature and, in the absence of acclimatization/adaptation, are likely to suffer large declines under mid-range International Panel for Climate Change predictions by 2050.