Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.     

Correlation of tumor oxygen dynamics with radiation response of the dunning prostate R3327-HI tumor

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO(2) measurements were performed on size-selected tumors several hours before radiotherapy using (19)F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm(3)) exhibited greater hypoxia than the smaller tumors (<2 cm(3); P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO(2) value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO(2), in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.     

Structure of the Rab7:REP-1 complex: insights into the mechanism of Rab prenylation and choroideremia disease

Members of the RabGDI/REP family serve as multifunctional regulators of the Rab family of GTP binding proteins. Mutations in members of this family, such as REP-1, lead to abnormalities, including progressive retinal degradation (choroideremia) in humans. The crystal structures of the REP-1 protein in complex with monoprenylated or C-terminally truncated Rab7 proteins revealed that Rab7 interacts with the Rab binding platform of REP-1 via an extended interface involving the Switch 1 and 2 regions. The C terminus of the REP-1 molecule functions as a mobile lid covering a conserved hydrophobic patch on the surface of REP-1 that in the complex coordinates the C terminus of Rab proteins. Using semisynthetic fluorescent Rab27A, we demonstrate that although Rab27A can be prenylated by REP-2, this reaction can be effectively inhibited by other Rab proteins, providing a possible explanation for the accumulation of unprenylated Rab27A in choroideremia.     

Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.     

Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole

Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.     

Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.     

Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. alpha-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure alpha-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified alpha-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both alpha-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of alpha-sarcin and made the lambda(em)m shift from 340 to 347 nm. Titration of alpha-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of alpha-sarcin molecule. This indicated that Trps were not involved in the binding of alpha-sarcin to chitin. Glycochitin in the culture medium increased the expression of alpha-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of alpha-sarcin and AFP, glycochitin increased the nuclease activity of alpha-sarcin.     

Marine phage genomics

Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.