Nonviral gene therapy targeting cardiovascular system

The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual's cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer.     

Mouse proteasomal ATPases Psmc3 and Psmc4: genomic organization and gene targeting

PSMC3 and PSMC4, components of the 19S complex of the 26S proteasome, show a significant degree of amino acid similarity, especially in the conserved ATPase domain (CAD). In this study, we characterized the mouse Psmc3 and Psmc4 genes. The genomic structures of both genes showed a significant degree of similarity. The Psmc3 gene was composed of 12 coding exons, whereas the Psmc4 gene had 11 exons. Exons encoding the leucine zipper domain and CAD were identical in number between the Psmc3 and Psmc4 genes. The Psmc3 gene mapped to mouse chromosome 2, whereas Psmc4 mapped to chromosome 7. We further addressed the biological roles of Psmc3 and Psmc4 through the generation of gene targeted mice. Both Psmc3- and Psmc4-deficient mice died before implantation, displaying defective blastocyst development. These findings indicate that Psmc3 and Psmc4 have similar and essential roles in early embryogenesis and further that both ATPases have noncompensatory functions in vivo.     

Roles of lysophosphatidic acid in cardiovascular physiology and disease

The bioactive lipid mediator lysophosphatidic acid (LPA) exerts a range of effects on the cardiovasculature that suggest a role in a variety of critical cardiovascular functions and clinically important cardiovascular diseases. LPA is an activator of platelets from a majority of human donors identifying a possible role as a regulator of acute thrombosis and platelet function in atherogenesis and vascular injury responses. Of particular interest in this context, LPA is an effective phenotypic modulator of vascular smooth muscle cells promoting the de-differentiation, proliferation and migration of these cells that are required for the development of intimal hyperplasia. Exogenous administration of LPA results in acute and systemic changes in blood pressure in different animal species, suggesting a role for LPA in both normal blood pressure regulation and hypertension. Advances in our understanding of the molecular machinery responsible for the synthesis, actions and inactivation of LPA now promise to provide the tools required to define the role of LPA in cardiovascular physiology and disease. In this review we discuss aspects of LPA signaling in the cardiovasculature focusing on recent advances and attempting to highlight presently unresolved issues and promising avenues for further investigation.     

在体细胞中实现基因打靶

体细胞基因打靶比较常见的ES细胞打靶是一项新发展的技术。本就如何设计选择打靶载体以及打靶适用的细胞系进行了较为详细的介绍。设计打靶的其他一些相关问题譬如顺序打靶、是否需要纯合DNA、高效的同源重组亦有讨论,从而对体细胞基因打靶特别是ES细胞基因打靶有一比较全面的了解。     

Site-specific gene targeting for gene expression in eukaryotes

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.     

A graphical simulation software for instruction in cardiovascular mechanics physiology

Background  

Computer supported, interactive e-learning systems are widely used in the teaching of physiology. However, the currently available complimentary software tools in the field of the physiology of cardiovascular mechanics have not yet been adapted to the latest systems software. Therefore, a simple-to-use replacement for undergraduate and graduate students' education was needed, including an up-to-date graphical software that is validated and field-tested.     

The role of antimicrobial peptides in cardiovascular physiology and disease

Antimicrobial peptides are natural peptide antibiotics, existing ubiquitously in both plant and animal kingdoms. They exhibit broad-spectrum antimicrobial activity and play an important role in host defense against invading microbes. Recently, these peptides have been shown to possess activities unrelated to direct microbial killing and be involved in the complex network of immune responses and inflammation. Thus, their role has now broadened beyond that of endogenous antibiotics. Because of their wide involvement in inflammatory response and the emerging role of inflammation in atherosclerosis, antimicrobial peptides have been proposed to represent an important link between inflammation and the pathogenesis of atherosclerotic cardiovascular diseases. This review highlights recent findings that support a role of these peptides in cardiovascular physiology and disease.     

IRAG and novel PKG targeting in the cardiovascular system

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1β isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1β and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.     

Re-engineering plant gene targeting

The genome sequence of Arabidopsis is complete and the genomes of plants representing legumes (Medicago truncatula) and grasses (rice) will soon follow. The rate at which new genes have been discovered has far outstripped the pace at which their function is determined. The greatest hurdle that plant biologists face in assigning gene function and in crop improvement is the lack of efficient and robust technologies to generate gene replacements or targeted gene knockouts. Many of the factors underlying these events remain to be elucidated. This review addresses the current status of plant gene targeting and what is known about the associated plant DNA repair mechanisms.     

Efficient ends-out gene targeting in Drosophila

In this report, we describe several approaches to improve the scalability and throughput of major genetic crosses in ends-out gene targeting. We generated new sets of targeting vectors and fly stocks and introduced a novel negative selection marker that drastically reduced the frequency of false-positive targeting candidates.     

PCR-based gene targeting in Candida albicans

PCR-based gene-targeting approaches have increased the speed of gene function analyses in ascomycetous fungi, for example, in the diploid human fungal pathogen Candida albicans. Here we describe a protocol that utilizes Rapid-PCR to amplify all cassettes available with the previously reported pFA modules. With this protocol, sufficient quantities of any cassette for use in C. albicans transformation experiments can be reliably generated in 25-50 min using either of the two alternative optimized amplification conditions; cassette amplification by standard PCR methods typically takes 3-4 h and is likely to require optimization of amplification conditions for each cassette. Transformants that appear 2-4 d after transformation can be rapidly identified using Rapid-PCR on whole cells, eliminating the need for genomic DNA extraction. In total, less than a week is required for the deletion of one allele in C. albicans. Repeating this procedure will result in the generation of homozygous mutant strains.     

Conditional gene targeting in the kidney

Complete mapping of the genome in a number of organisms provides a challenge for experimental nephrologists to identify potential functions of a vast number of new genes in the kidney. Since knockout technologies have evolved in the early eighties the mouse has become a valuable model organism. Researchers can now artificially eliminate the expression of specific genes in a mammalian organism and examine the phenotype. New developments have emerged that allow investigators to knock out a gene specifically in the kidney. Several kidney-specific promoters provide valuable tools and bacterial artificial chromosome (BAC) based techniques like recombineering will enhance both number and accuracy of new mouse lines with spatially controlled gene expression. In addition to spatial control, tetracycline- or tamoxifen-inducible systems, provide the possibility of influencing the temporal expression pattern of a gene enabling researchers to dissect its functions in adult organisms. Knocking out a gene will continue to be the gold standard for defining the role of a specific gene whereas tissue-specific gene knockdown using RNA interference represents an alternative approach for generating lower-priced and fast loss of function models. In addition to reverse genetic approaches, forward genetic techniques like random mutagenesis in mice continue to evolve and will enhance our understanding of disease mechanisms in the kidney.     

Role of thyroid hormones-induced oxidative stress on cardiovascular physiology

Thyroid hormones (THs) play an essential role in the maintenance of cardiovascular homeostasis and are involved in the modulation of cardiac contractility, heart rate, diastolic function, systemic vascular resistance, and vasodilation. THs have actions on cardiovascular physiology through the activation or repression of target genes or the activation of intracellular signals through non-genomic mechanisms. Hyperthyroidism alters certain intracellular pathways involved in the preservation of the structure and functionality of the heart, causing relevant cardiovascular disorders. Reactive oxygen species (ROS) play an important role in the cardiovascular system, but the exacerbated increase in ROS caused by chronic hyperthyroidism together with regulation on the antioxidant system have been associated with the development of cardiovascular dysfunction. In this review, we analyze the role of THs-induced oxidative stress in the cellular and molecular changes that lead to cardiac dysfunction, as well as the effectiveness of antioxidant treatments in attenuating cardiac abnormalities developed during hyperthyroidism.     

Function and regulation of mitofusin 2 in cardiovascular physiology and pathology

Mitochondrial dynamics with constant fusion and fission plays vital roles in regulating cellular biological processes. Mitofusin 2 (Mfn2) is dynamin-related protein whose activity promotes mitochondrial fusion and maintains the homeostasis of mitochondrial dynamics. Advanced studies have demonstrated that Mfn2 is a multifunctional protein with signaling roles beyond fusion. Mfn2 is actively involved in various biological processes under both physical and pathological conditions, including mitochondrial transport and the interaction between endoplasmic reticulum/sarcoplasmic reticulum and mitochondria, as well as cell metabolism, apoptosis and autophagy. This review summarises the structural and functional properties of Mfn2, with focus on recent advances in its regulatory role in cardiovascular system.     

Homologous recombination and gene targeting

Gene therapy and the production of mutated cell lines or model animals both require the development of efficient, controlled gene-targeting strategies. Classical approaches are based on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. The low frequency of homologous recombination in mammalian cells leads to inefficient targeting. Here, we review the limiting steps of classical approaches and the new strategies developed to improve the efficiency of homologous recombination in gene-targeting experiments.