Biomedical subjects

James Lausier

Publications and source records attributed to James Lausier.

6 recordsLinked to original sources

PubMed2006

Regulation of pancreatic beta-cell regeneration in the normoglycemic 60% partial-pancreatectomy mouse.

beta-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory beta-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was approximately 68% of the shams by 15 days, partly due to enhanced beta-cell proliferation. A transient approximately 2.8-fold increase in the prevalence of beta-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory beta-cell growth by activating beta-cell proliferation with an increase in the number of beta-cell clusters/small islets.

Actins↗

PubMed2005

Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase.

The physiological mechanisms underlying the compensatory growth of beta-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of beta-cell mass. A 3.8-fold beta-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened beta-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of beta-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased beta-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive beta-cell growth. During this phase, we also detected an increase in the numbers of small beta-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened beta-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in beta-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of beta-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.

Aging↗

PubMed2004

Gene delivery to respiratory epithelial cells by magnetofection.

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.

Animals↗

PubMed2004

Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer.

BACKGROUND: Gene delivery by the use of magnetic forces, so-called magnetofection, has been shown to enhance transfection efficiency of viral and non-viral systems up to several-hundred-fold. For this purpose gene carriers, such as polyethylenimine (PEI), are associated with superparamagnetic nanoparticles and complexed with plasmid DNA. Gene delivery is targeted by the application of a magnetic field. METHODS: To investigate the underlying mechanism, we studied the impact of the applied magnetic field on the transfection process of PEI-coated superparamagnetic iron oxide gene vectors (magnetofectins) using various cell lines. In particular, we addressed the question whether accelerated sedimentation of magnetofectins is the driving force or if the magnetic field itself directly influences the endocytic processing of the magnetofectins. The cellular uptake mechanism of magnetofectins was studied by electron microscopy and transfection experiments in the presence of various inhibitors that operate at different steps of endocytosis. RESULTS: In this study we could show that cellular uptake of magnetofectins proceeds obviously by endocytosis. Cellular uptake of magnetofectins behaves almost analogously as compared with PEI polyplexes. Besides unspecific endocytosis, apparently clathrin-dependent as well as caveolae-mediated endocytic uptake is involved. CONCLUSIONS: The magnetic field itself does not alter the uptake mechanism of magnetofectins. Obviously, the magnetic forces lead to an accelerated sedimentation of magnetofectins on the cell surface and do not directly affect the endocytic uptake mechanism. So further improvement of magnetic field application could lead to efficient targeting of gene expression into the desired organ and tissue in vivo.

Antimycin A↗

PubMed2003

A novel transfecting peptide comprising a tetrameric nuclear localization sequence.

The transport of exogenous DNA into the nucleus of eukaryotic cells is a prerequisite for successful gene delivery. To favor nuclear transport we synthesized a tetramer of the nuclear localization signal (NLS) of the SV40 large T-antigen as a novel nonviral gene delivery vector. This 4.4-kDa lysine-rich peptide (NLSV404) binds and compacts DNA by electrostatic interaction and forms stable polyplexes. Apart from its sequence-specific potency to mediate nuclear accumulation of conjugated albumin, NLSV404 also displays properties of nuclear transport for plasmid DNA as confirmed by fluorescence in situ hybridization. Further, NLSV404 polyplexes are shown to efficiently transfect various cell lines such as 16HBE14o-, HeLa S6, and Cos7 cells. NLSV404 polyplexes displayed at least 20-fold higher transfection rates than analogous polyplexes formed by the nuclear transport-deficient mutant sequence cNLS. Using growth-arrested cells, NLSV404 complexes were at least 100-fold more efficient than cNLS complexes. Combination of NLSV404 peptide but not of cNLS peptide with preformed polyethylenimine and dendrimer DNA complexes resulted in a strong increase in transfection efficiency. Incubation of cells prior to transfection with NLSV404 polyplexes with excess free peptide NLSV404 but not with cNLS resulted in a dose-dependent dramatic decrease in the transfection rate, suggesting a sequence-specific competitive inhibition. These results indicate that NLSV404 mediates nuclear accumulation of transfected plasmid DNA and that it can be a highly useful component of nonviral gene vectors.

Active Transport, Cell Nucleus↗

PubMed2003

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells.

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.

Amino Acid Motifs↗