An international database and integrated analysis tools for the study of cancer gene expression.

Researchers working collaboratively in Brazil and the United States have assembled an International Database of Cancer Gene Expression. Several strategies have been employed to generate gene expression data including expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and open reading-frame expressed sequence tags (ORESTES). The database contains six million gene tags that reflect the gene expression profiles in a wide variety of cancerous tissues and their normal counterparts. All sequences are deposited in the public databases, GenBank and SAGEmap. A suite of informatics tools was designed to facilitate in silico analysis of the gene expression datasets and are available through the NCI Cancer Genome Anatomy Project web site (http://cgap.nci.nih.gov).

In silico analysis of cancer through the Cancer Genome Anatomy Project.

The Cancer Genome Anatomy Project (CGAP) was designed and implemented to provide public datasets, material resources and informatics tools to serve as a platform to support the elucidation of the molecular signatures of cancer. This overview of CGAP describes the status of this effort to develop resources based on gene expression, polymorphism identification and chromosome aberrations, and we describe a variety of analytical tools designed to facilitate in silico analysis of these datasets.

Tight insertion of cytochrome b5 into large unilamellar vesicles.

Cytochrome b5 spontaneously binds to liposomes in a 'loose', or transferable form, whereas in vivo b5 binds post-translationally to the ER in the 'tight' or nontransferable form. The mechanism of tight insertion is unknown, except that it does not require SRP or energy input. The present study shows that prolonged incubation of b5 with large unilamellar vesicles (LUVs) of phosphatidylcholine results in slow conversion of the loose to the tight form, with a halftime of days. However, the process is complex. When the b5-LUVs are depleted of loose b5, by transfer of b5 to sonicated vesicles, the tight b5 is found to be concentrated to near saturating levels in a small fraction of the LUVs. If the LUVs devoid of tight b5 are recovered and then reincubated with fresh b5, the same slow transformation recurs. Apparently, a new population of vesicles, containing tight b5, is generated during the prolonged incubation with the protein. The b5-enriched LUVs contain about the same level of trapped sucrose as does the original vesicle preparation, indicating that vesicle integrity is maintained throughout the process. When fresh b5 is added to these tight b5-containing LUVs, all the freshly bound protein rapidly inserts (< 2 h) into the tight configuration. Apparently, the newly formed tight-b5/LUV vesicle population is 'insertion-active'. A model for these complex transformations is proposed.

Transbilayer migration (flip-flop) of 12-(4-azido-2-nitrophenoxy)stearoyl glucosamine in large unilamellar phospholipid vesicles.

The ability of the glycolipid photoprobe, 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (12-APS-GlcN), to undergo transbilayer flip-flop and intermembrane transfer between liposomes was examined. It was found that probe which was incorporated into membranes during the preparation of large unilamellar vesicles (LUVs) could be rapidly and completely extracted by incubation of these donor vesicles (in the liquid-crystalline state) with probe-free acceptor vesicles.

Distribution of cytochrome b5 between small and large unilamellar phospholipid vesicles.

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose" or transferable configuration, characterized by the ability of the protein to rapidly equilibrate between vesicles. In a preliminary report we showed that the distribution of cytochrome b5 among a heterogeneous population of small sonicated phosphatidylcholine vesicles (212 to about 350 A in diameter) lies in favor of the smallest vesicles by a factor of at least 20 (Greenhut, S.F. and Roseman, M.A. (1985) J. Biol. Chem. 260, 5883-5886). In the present studies we have attempted to determine the maximal extent to which bilayer curvature can influence the intervesicle distribution of cytochrome b5, by measuring the distribution of the protein between a population of limit-size vesicles 212 A in diameter and a population of large unilamellar vesicles approximately 1000 A in diameter. (The effect of bilayer curvature on the physical properties of the lipids in the large vesicles is considered to be negligible.) The results show that cytochrome b5 favors the small vesicle population by a factor of about 200. This observation suggests that the formation of highly curved regions in biological membranes (or the formation of regions in which the physical state of the lipids is similar to that in small vesicles) may cause the accumulation of certain membrane proteins at those sites. We also observed that a significant fraction (11-20%) of the cytochrome b5, when added directly to the large vesicles, spontaneously inserts into the "tight," physiologically proper configuration. A possible mechanism is discussed.

Distribution of cytochrome b5 between sonicated phospholipid vesicles of different size.

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose," or transferable, configuration characterized by the ability of the protein to rapidly equilibrate between vesicles. A heterogeneous dispersion of sonicated phosphatidylcholine vesicles, 212 to about 350 A in diameter, was prepared by differential centrifugation. When cytochrome b5 was incubated with these vesicles (1 mol of protein/833 mol of phospholipid, in 0.01 M NaHCO3, 0.1 M NaCl, 10(-4) M EDTA, pH 7.4) and the mixture was subjected to molecular sieve chromatography on Sepharose 2B-CL, the cytochrome b5 was found to elute preferentially with the smaller vesicles. Subsequently, a fresh preparation of heterogeneous vesicles was subfractionated by gel filtration, and the individual fractions were incubated with the protein. Molecular sieve chromatography of these complexes showed that cytochrome b5 favors the smallest over the largest vesicles by a factor of at least 20. This result suggests that formation of highly curved regions in biological membranes may cause accumulation of certain membrane proteins at those sites.

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles.

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.