DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis

Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.     

Enhanced cellular uptake with a cobaltacarborane-porphyrin-HIV-1 Tat 48-60 conjugate

A series of four porphyrin-cobaltacarborane conjugates have been synthesized, containing three or four cobaltabisdicarbollide anions linked by O(CH(2)CH(2)O)(2) groups to the porphyrin macrocycle and one of them containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity, and preferential sites of intracellular localization of the conjugates were evaluated in human HEp2 cells. All conjugates are nontoxic in the dark at the concentrations studied. Upon exposure to low light dose (1 J cm(-)(2)) only the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate showed 30% inhibition of cell proliferation at a concentration of 10 microM. The cellular uptake was dependent on the number of carborane cages and was significantly enhanced by the presence of the cell penetrating peptide sequence HIV-1 Tat 48-60. All conjugates preferentially localized in the cell lysosomes.     

Scanning cytometry with a LEAP: laser-enabled analysis and processing of live cells in situ.

BACKGROUND: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). METHODS: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. RESULTS: Live cell samples (adherent and suspension) could be purified to 90-100% purity with 50-90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3-70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5-60 microm to targeted areas under conditions used here. CONCLUSIONS: LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short.     

Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives

Inhibitors targeting pancreatic alpha-amylase and intestinal alpha-glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase-glucoamylase (MGA), a family 31 glycoside hydrolase, is an alpha-glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective alpha-glucosidase inhibitors modeled after salacinol, a naturally occurring alpha-glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five-membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six-membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity.     

Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen

Secreted and cell surface proteins play essential roles in numerous essential biological processes in eukaryotic organisms, but are often more difficult to isolate and identify than proteins that are localized in intracellular compartments. However, several high-throughput 'gene-trap' techniques have been developed to characterize these 'secretomes', including the yeast secretion trap (YST) screen. This method involves fusing cDNA libraries from the tissue or cell type of interest to a yeast (Saccharomyces cerevisiae) invertase reporter gene, transforming the resulting fusion library into an invertase-deficient yeast strain and plating the transformants on a medium containing sucrose as the sole carbon source. A yeast cell with a transgene encoding a secreted or cell surface protein can synthesize a secreted invertase fusion protein that can rescue the mutant, and the plasmid DNA can then be sequenced to identify the gene that encodes it. We describe a recently improved version of this screen, which allows the identification of genes encoding secreted proteins in 1-2 months.     

Weekly changes in basal metabolic rate with eight weeks of overfeeding

Objective : The contribution of basal metabolic rate (BMR) to weight gain susceptibility has long been debated. We wanted to examine whether BMR changes in a linear fashion with overfeeding. Our hypothesis was that BMR does not increase linearly with 1000‐kcal/d overfeeding in lean healthy subjects over 8 weeks. The null hypothesis states that BMR increases linearly with 1000‐kcal/d overfeeding in lean healthy subjects. Research Methods and Procedures : Initially, 16 lean healthy sedentary subjects completed 2 weeks of weight maintenance feeding at the General Clinical Research Center. The subjects were then overfed by 1000 kcal/d over 8 weeks. BMR was measured under standard conditions each week using indirect calorimetry. Results : Baseline BMR was 1693 ± 154.5 kcal/d. BMR increased from 1711 ± 201.3 kcal/d at week 1 of overfeeding to 1781 ± 171.65 kcal/d at the second week of overfeeding (p = 0.05). BMR fell during the third week of overfeeding to 1729 ± 179.5 kcal/d (p = 0.05). After 5 weeks of overfeeding, BMR reached a plateau. Thereafter, there was no further change. Comparison of BMR with weeks of overfeeding was significantly different compared with the linear model (p < 0.05). Discussion : Increases in BMR in lean sedentary healthy subjects with 1000‐kcal/d overfeeding are not linear over 8 weeks. There seems to be a short‐term increase in BMR in the first 2 weeks of overfeeding that is not representative of longer‐term changes.     

Habituation to environmental enrichment in captive sloth bears—effect on stereotypies

The benefits to captive animals of environmental enrichment (EE) are widely recognized. Few studies have, however, studied how to maximise the effect of EE. One issue with EE programs seems to be habituation to the enrichment device. To study the effect of habituation to EE, 14 captive sloth bears (Melursus ursinus) were subjected to two different EE treatments. Treatment one presented EE (logs with honey containing holes) for five consecutive days, whereas treatment two presented EE on intermittent days for five days. Intermittent presentations tended to reduce habituation toward the EE. Both consecutive and intermittent presentations significantly reduced stereotypies; however, the consecutive presentations had a longer‐lasting effect. Explorative behaviors increased in both treatments, consistent with earlier findings that EE increase levels of natural behaviors. Other behaviors were unaffected by the EE presentations. The results show that intermittent presentation of EE objects may secure the interest of the animals, but continuous access to enrichment may be more efficient in reducing stereotypies in the long run. Zoo Biol 29:705–714, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of a biologically active isomer of kotalanol,a naturally occurring glucosidase inhibitor

The syntheses of an isomer of kotalanol, a naturally occurring glucosidase inhibitor, and of kotalanol itself are described. The target compounds were synthesized by nucleophilic attack of PMB-protected 1,4-anhydro-4-thio-d-arabinitol at the least hindered carbon atom of two 1,3-cyclic sulfates, which were synthesized from d-mannose. Methoxymethyl ether and isopropylidene were chosen as protecting groups. The latter group was critical to ensure the facile deprotection of the coupled products in a one-step sequence to yield kotalanol and its isomer. The stereoisomer of kotalanol, with the opposite stereochemistry at the C-6′ stereogenic centre, inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K_i value of 0.20 ± 0.02 μM; this compares to a K_i value for kotalanol of 0.19 ± 0.03 μM. The results indicate that the configuration at C-6′ is inconsequential for inhibitory activity against this enzyme.     

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.