Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells.

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer (M(r) 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the headgroup (referred to as CPL(1)-CPL(4), respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios < or =0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60 degrees C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca(2+). The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL(2) displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL(3), while for CPL(4)-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.

Fluorescent-labeled poly(ethylene glycol) lipid conjugates with distal cationic headgroups.

The synthesis of a new class of fluorescent cationic poly(ethylene glycol) lipid conjugates (CPLs) is described. These lipids consist of a hydrophobic distearoyl-phosphatidylethanolamine (DSPE) anchor coupled to a highly fluorescent N(epsilon)-dansyl lysine moiety, which is attached to a hydrophilic poly(ethylene glycol) (PEG) spacer that is linked to a cationic headgroup made of lysine residues. Introduction of the dansyl moiety allows rapid and accurate quantification of CPLs within lipid bilayers using fluorescence techniques. The synthetic scheme is straightforward, using repeated amino-carboxyl coupling reaction steps, with purification by precipitation. A series of dansylated CPLs was synthesized with zero, one, three, and seven lysine residues located at the distal end of the PEG chain, giving rise to CPLs with one, two, four, and eight distal positive charges, respectively. The structures of the CPLs were confirmed by (1)H NMR spectroscopy and chemical analysis. CPLs provide a means of introducing positive charge to a bilayer that is localized some distance from the membrane surface, and are of particular interest for nonviral gene delivery applications. The usefulness of CPLs is demonstrated by the enhanced in vitro cellular binding and uptake of liposomes containing CPL(4).

Implementing problem-based learning in a family medicine clerkship.

BACKGROUND AND OBJECTIVES: Problem-based learning (PBL) has been implemented in the curriculum of many medical schools, but limited information is available about the outcome of this learning technique. The educational intervention presented in this paper implemented a PBL learning component in our third-year family medicine clerkship and measured the outcomes of this curricular change. METHODS: One third of the curricular time devoted to didactic teaching in our family medicine clerkship was replaced with PBL activities. Simulated cases were developed and presented to students who, with the aid of faculty facilitators, studied the cases, gathered information about the cases, and developed diagnostic and management plans for the cases. The outcome of the intervention was measured by a) comparing students' scores on the National Board of Medical Examiners (NBME) family medicine clerkship examination to scores achieved by students in the year before PBL was introduced and b) students' evaluations of the relevance and success of PBL in the clerkship curriculum. RESULTS: Students' NBME clerkship examination scores increased from a mean of 66 the year before PBL began to 73 after PBL was implemented. More than 80% of students reported that PBL was a good way to learn family medicine, and 85% reported that the PBL technique provided sufficient information to formulate learning issues. CONCLUSIONS: PBL can be introduced into a third-year family medicine clerkship curriculum with general acceptance by students. Students rated the technique highly, and their examination scores improved.

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.

Mapping the membrane topology of the closed state of the colicin E1 channel.

The membrane-associated closed channel state of the colicin E1 thermolytic peptide was studied by the Parallax Method of depth-dependent fluorescence quenching. A number of single Trp-containing peptides of colicin E1 were prepared to facilitate the use of Trp as a probe for the topography of the channel peptide in the membrane-bound state. The bound form of the channel peptide was studied by binding channel peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-pal-mitoyl -2-oleoyl-sn- glycero-3-phosphatidylglycerol large unilamellar vesicles (60:40, mol/mol, approximately 0.1 microns diameter vesicles prepared by an extrusion technique) at low pH (pH 3.5). Depth-dependent fluorescence quenching studies using two nitroxide-labeled phospho-lipids (1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphatidylcho++ +-line and 1-palmitoyl-2-(12-doxylstearoyl)-sn-glycero-3-phosphatidylcholine) were conducted to determine the membrane location of each Trp residue for the vesicle-bound peptide. The three naturally occurring Trp residues in the colicin channel peptide, Trp-424, Trp-460, and Trp-495, were found to reside at membrane depths (from the C-2 carbon of the fatty acyl chain) of 7.4, 3.1, and 8.4 A, respectively. Three Trp residues (Trp-355, Trp-460, and Trp-507) in the channel peptide were classified as shallow (0-5.0 A from C-2 carbon). The remaining 9 Trp residues were classified as moderately buried (5.1-10.0 A). None of the dozen tryptophyls were classified as deeply buried in the membrane bilayer (10.1-15.0 A). A model for the colicin E1 channel based on these measurements along with previous data obtained from proteolysis, chemical labeling, ESR quenching, and mutagenesis experiments is proposed. This model for the closed state of the channel has as its central feature of the presence of only two trans-membrane segments. The membrane-associated portion of the channel includes the hydrophobic membrane anchor domain, Ala-474 to Ile-508. Furthermore, the fluorescence quenching data are consistent with the NH2-terminal helices (helices 1-7) lying on the surface of the membrane with the helical axis being oriented parallel to the membrane plane.

Acrylamide quenching of the intrinsic fluorescence of tryptophan residues genetically engineered into the soluble colicin E1 channel peptide. Structural characterization of the insertion-competent state.

Colicin E1 or any of its COOH-terminal channel peptides can be activated in vitro by acidic (< 4.5) pH or detergents. In its activated or insertion-competent state, the colicin E1 thermolytic (178 residue) channel peptide demonstrated an increased ability to bind and form channels in artificial membranes. An earlier report [Merrill et al. (1990) Biochemistry 29, 5829-5836] indicated that the structural change occurring in the channel peptide upon activation was not a large unfolding but seemingly involves a more subtle conformational change. To probe the solution structure of the colicin channel peptide and the structural changes occurring upon activation, 12 single-tryptophan-containing mutant peptides have been prepared. All of the peptides displayed cellular cytotoxicity comparable to the wild-type peptide. Fluorescence quenching by acrylamide of each Trp residue genetically engineered into the channel peptide indicated that tryptophyls located at positions 355, 367, 393, 413, and 443 report significant conformational changes which are associated with the insertion-competent state. Calculation of the bimolecular quenching constants for each single-Trp peptide showed that there are three classes of Trp residues found in the native colicin E1 channel peptide. None of the Trp residues were found to be completely inaccessible to acrylamide (buried). The NH2-terminal region near Trp-355 and -367 along with the COOH-terminal hydrophobic domain, including Trp-484, -495, and -507, was largely buried in the channel peptide soluble structure. Two peptide segments, one containing Trp-393, -404, and -413 and a second encompassing Trp-431 and -443, were moderately to very exposed regions in the soluble channel peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Leucemia monoblástica aguda: santuario cutáneo / Acute monoblastic leukemia: cutaneous santuaries

Presentamos una leucemia monoblástica aguda con múltiples infiltrados dérmicos específicos iniciales rebeldes a la quimioterapia, hematológicamente eficaz. Insistimos en la necesidad de diagnosticar (impronta) y evaluar la magnitud de los mismos con fines diagnósticos (biopsia), así como en la detección de posibles santuarios cutáneos, para lo cual la inmunomarcación de muramidasa resultó de gran valor