Gene delivery by cationic lipid vectors: overcoming cellular barriers

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

An investigation of <Emphasis Type="Italic">Clostridium</Emphasis> species present in nutraceutical preparations of <Emphasis Type="Italic">Arthrospira platensis</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) for human consumption

The presence of the anaerobic spore former Clostridium in Arthrospira platensis destined for human consumption is generally not assessed during quality assurance procedures. As this nutraceutical is administered as complementary medicine to the immunocompromised, this study aimed to investigate the presence of these potential pathogens. Anaerobic counts performed on tablets from a single manufacturer indicated an excess of 10⁵ CFU/endospores g⁻¹ tablet for three different A. platensis batches. Tests for coliforms for use as “indicators” of pathogens in the tablets were negative. Using classic culture techniques, five species of Clostridium were isolated. Subsequent use of PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting of tablets showed a divergent microbial population, with a predominance of anaerobic endospore formers, including Clostridium. Sequencing of a 1.5 kb 16S rDNA clone library and phylogenetic analyses of prominent operational taxonomic units confirmed the presence of an additional five Clostridium spp. and other genera in the tablets. A composite molecular ladder, using 16S rRNA DGGE amplicons of 17 representative bacterial species was constructed to assist in identifying anaerobes present in tablets sourced from three different A. platensis manufacturers. Results indicated that commercial A. platensis preparations were contaminated with potentially hazardous clostridia and other anaerobic species. Results suggest that certain commercial A. platensis preparations require stringent microbial quality assurance measures to ensure safe use as a nutraceutical for the immunocompromised and the general public.     

Initial mutations direct alternative pathways of protein evolution

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.     

Cationic lipid-mediated transfection in vitro and in vivo (review).

Recent rapid developments in genomics will likely lead to a rapid expansion in identifying defective genes causing a variety of diseases, implying a vast increase in the number of therapeutic targets. Treatment of such diseases may include strategies ranging from gene delivery and replacement to antisense approaches. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene- or oligonucleotide-carrier systems, which are necessary for protective purposes (against nucleases) and transport (to target tissue and cells in vivo). Further, they should also display the propensity to efficiently translocate the oligonucleotides and gene constructs into cells, via passage across several membrane barriers. The emphasis in this review will be on the use of cationic lipids for that purpose. Crucial to successful application of this sophisticated technology in vivo will be a need for a better understanding of fundamental and structural parameters that govern transfection efficiency, including the issues of cationic lipid/DNA complex assembly (with or without helper lipid), stability towards biological fluids, complex-target membrane interaction and translocation, and gene-integration into the nucleus. Biophysical and biochemical characterization of so-called lipoplexes, and their interaction with cells in vitro, are considered instrumental in reaching such insight. Here, most recent advances in cationic lipid-mediated gene delivery are discussed from such a perspective.     

Comparing artificial and natural selection in rate of adaptation to genetic stress in Aspergillus nidulans

In an experimental study of adaptation to negative pleiotropic effects of a major fungicide resistance mutation in the filamentous fungus Aspergillus nidulans we have investigated the relative effectiveness of artificial selection vs. natural selection on the rate of compensatory evolution. Using mycelial growth rate as a fitness measure, artificial selection involved the weekly transfer of the fastest growing sector onto a fresh plate. Natural selection was approximated by transferring random samples of all the spores produced by the mycelium. Fungicide resistant and fungicide sensitive haploid and diploid strains were used in an evolution experiment over 10 weekly transfers, which is equivalent to 1200 cell cycles. Two different environmental conditions were applied: a constant fungicide-free environment and a weekly alternation between presence and absence of fungicide. Results show that for all strains and conditions used the transfer of a random sample of all spores leads to more rapid adaptation than the transfer of the visually 'fittest' sector. The rates of compensatory evolution in the constant and the alternating environment did not differ. Moreover, haploid strains tend to have a higher rate of adaptation than isogenic diploid strains.     

Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis

PURPOSE OF REVIEW: The appearance of scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in macrophages and liver implicates these transporters in different stages of reverse cholesterol transport. This review focuses on the role of SR-BI and ABCA1 in reverse cholesterol transport in the context of atherosclerotic lesion development. RECENT FINDINGS: Recent studies indicate that hepatic expression of ABCA1 and SR-BI is important for the generation of nascent HDL and the delivery of HDL cholesteryl esters to the liver, respectively. Although macrophage SR-BI and ABCA1 do not contribute significantly to circulating HDL levels, the perpetual cycle of HDL lipidation and delipidation by the liver ensures the availability of acceptors for cholesterol efflux that maintain cholesterol homeostasis in arterial macrophages, thereby reducing atherogenesis. In addition to its established role in the selective uptake of HDL cholesteryl esters, there is now evidence that hepatic SR-BI facilitates postprandial lipid metabolism, and that hepatic secretion of VLDL is dependent on ABCA1-mediated nascent HDL formation. Thus, remnant and HDL metabolism are more intimately intertwined in hepatic lipid metabolism than has previously been appreciated. SUMMARY: Recent advances in the understanding of the role of ABCA1 and SR-BI in HDL metabolism and their atheroprotective properties indicate the significant potential of modulating ABCA1 and SR-BI expression in both arterial wall macrophages and the liver for the treatment of atherosclerotic coronary artery disease.     

Neurogenetic interactions and aberrant behavioral co-morbidity of attention deficit hyperactivity disorder (ADHD): dispelling myths

Background

Attention Deficit Hyperactivity Disorder, commonly referred to as ADHD, is a common, complex, predominately genetic but highly treatable disorder, which in its more severe form has such a profound effect on brain function that every aspect of the life of an affected individual may be permanently compromised. Despite the broad base of scientific investigation over the past 50 years supporting this statement, there are still many misconceptions about ADHD. These include believing the disorder does not exist, that all children have symptoms of ADHD, that if it does exist it is grossly over-diagnosed and over-treated, and that the treatment is dangerous and leads to a propensity to drug addiction. Since most misconceptions contain elements of truth, where does the reality lie?

Results

We have reviewed the literature to evaluate some of the claims and counter-claims. The evidence suggests that ADHD is primarily a polygenic disorder involving at least 50 genes, including those encoding enzymes of neurotransmitter metabolism, neurotransmitter transporters and receptors. Because of its polygenic nature, ADHD is often accompanied by other behavioral abnormalities. It is present in adults as well as children, but in itself it does not necessarily impair function in adult life; associated disorders, however, may do so. A range of treatment options is reviewed and the mechanisms responsible for the efficacy of standard drug treatments are considered.

Conclusion

The genes so far implicated in ADHD account for only part of the total picture. Identification of the remaining genes and characterization of their interactions is likely to establish ADHD firmly as a biological disorder and to lead to better methods of diagnosis and treatment.

     

Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of HDL cholesteryl esters is well established. In SR-BI-deficient mice, we recently observed a delayed postprandial triglyceride (TG) response, suggesting an additional role for SR-BI in facilitating chylomicron (CM) metabolism. Here, we assessed the effect of adenovirus-mediated hepatic overexpression of SR-BI (Ad.SR-BI) in C57BL/6J mice on serum lipids and CM metabolism. Infection of 5 x 10(8) plaque-forming units per mouse of Ad.SR-BI significantly decreases serum cholesterol (>90%), phospholipids (>90%), and TG levels (50%), accompanied by a 41.4% reduction (P < 0.01) in apolipoprotein B-100 levels. The postprandial TG response is 2-fold lower in mice treated with Ad.SR-BI compared with control mice (area under the curve = 31.4 +/- 2.4 versus 17.7 +/- 3.2; P < 0.05). Hepatic mRNA expression levels of genes known to be involved in serum cholesterol and TG clearance are unchanged and thus could not account for the decreased plasma TG levels and the change in postprandial response. We conclude that overexpression of SR-BI accelerates CM metabolism, possibly by mediating the initial capture of CM remnants by the liver, whereby the subsequent internalization can be exerted by additional receptor systems such as the LDL receptor (LDLr) and LDLr-related protein 1.