Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells.     

Two computer programs for rapid entry of DNA sequence data

Two computer programs for the IBM personal computer are describedfor rapid and accurate entry of DNA sequence data. The DNA sequencefiles produced can be used directly by the DNA sequence manipulationprograms by R. Staden (the DataBase system), the Universityof Wisconsin Genetics Computer Group, DNASTAR, or D. Mount.The first program, DIGISEQ, utilizes a sonic digitizer for semi-automationof sequence entry. To enter the DNA sequence each band of agel reading is touched by the stylus of the sonic digitizer.DIGISEQ corrects for both changes in lane width and lane curvature.The algorithm is extremely efficient and rarely requires re-entenngthe centers of the lanes. The second program, TYPESEQ, usesonly the keyboard for input. The keyboard is reconfigured toplace nucleotides and ambiguity codes under the fingers of onehand, corresponding to the order of the nucleotides on the geldefined by the user Both programs produce individual tones foreach nucleotide, and certain ambiguity codes. This verifiesinput of the correct nucleotide or ambiguity code, and thuseliminates the need to visually check the screen display duringsequence entry. Received on November 16, 1986; accepted on June 16, 1987     

Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser(756) of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.     

Failure to induce alopecia areata in C3H/HeJ mice with exogenous interferon gamma

Alopecia areata is a cell mediated autoimmune disease that targets actively growing, anagen stage hair follicles in several mammalian species. Upregulation of MHC I due to interferon gamma is considered to be one of the initiating steps. To test this hypothesis we used the spontaneous C3H/HeJ mouse model, induced anagen by wax stripping the skin, and injected recombinant murine interferon gamma. Alopecia areata is a complex polygenic trait with low penetrance in these mice. Injection of interferon gamma did not change the frequency or time of onset of alopecia in these mice suggesting this protein alone is not sufficient to initiate disease.     

Phenylpiperidine-benzoxazinones as urotensin-II receptor antagonists: synthesis, SAR, and in vivo assessment

Various 4-phenylpiperidine-benzoxazin-3-ones were synthesized and biologically evaluated as urotensin-II (U-II) receptor antagonists. Compound 12i was identified from in vitro evaluation as a low nanomolar antagonist against both rat and human U-II receptors. This compound showed in vivo efficacy in reversing the ear-flush response induced by U-II in rats.     

Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.     

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.     

Phagocytosis, an alternative model system for the study of cell adhesion

Cell adhesion encompasses a variety of cell-cell and cell-matrix adhesive interactions. Whereas ligation of most adhesion receptors activate Rho-family GTP-binding proteins and the subsequent reorganization of the actin cytoskeleton, the molecular mechanisms involved remain poorly understood. Because phagocytosis is a spatially restricted adhesion process, it represents a simplified model system to investigate the spatio-temporal regulation of the signalling pathways that link surface adhesion receptors, small GTPases and the actin cytoskeleton. This review highlights some of the similarities between the formation and maintenance of adhesive contacts and phagocytic uptake and discusses why the study of phagocytosis can help understand more complex adhesion processes.     

An efficient method for routine epstein-barr virus immortalization of human B lymphocytes

Summary A variety of methods exist for the immortalization of B lymphocytes by Epstein-Barr virus due to the simplicity of such techniques to establish cell lines with stable genomic DNA. Two different methods for immortalizing lymphoblastoid cell lines were compared for differences in techniques and materials, time between initiation and immortalization, and success rate of immortalization. An incubation period in Epstein-Barr virus and the use of conditioned media improved immortalization efficiency from 86 to 98% and decreased the time (usually weeks) from culture initiation to cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in non-insulin-dependent diabetes mellitus.     

Calcium exchange and structural changes during the photosynthetic oxygen evolving cycle

PSII catalyzes the oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. PSII contains an oxygen-evolving complex, which is located on the lumenal side of the PSII reaction center and which contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen. This process produces five Sn-states, where n refers to the number of oxidizing equivalents stored. Calcium is required for oxygen production. Strontium is the only divalent cation that replaces calcium and maintains activity. In our previous FT-IR work, we assessed the effect of strontium substitution on substrate-limited PSII preparations, which were inhibited at the S3 to S0 transition. In this work, we report reaction-induced FT-IR studies of hydrated PSII preparations, which undergo the full S-state cycle. The observed difference FT-IR spectra reflect long-lived photoinduced conformational changes in the oxygen-evolving complex; strontium exchange identifies vibrational bands sensitive to substitutions at the calcium site. During the S1' to S2' transition, the data are consistent with an electrostatic or structural perturbation of the calcium site. During the S3' to S0' and S0' to S1' transitions, the data are consistent with a perturbation of a hydrogen bonding network, which contains calcium, water, and peptide carbonyl groups. To explain our data, persistent shifts in divalent cation coordination must occur when strontium is substituted for calcium. A modified S-state model is proposed to explain these results and results in the literature.