Adenovirus‐mediated introduction of DNA into pig sperm and offspring

The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested. Exposure of spermatozoa to adenovirus bearing the E. coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa. Treatment did not affect either viability or acrosomal integrity of boar sperm. Of the 2‐ to 8‐cell embryos obtained after in vitro fertilization with adenovirus‐exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus‐exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis. RT‐PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested. The offspring obtained after mating two positive animals did not show LacZ gene presence. Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation. Mol. Reprod. Dev. 53:149–158, 1999. © 1999 Wiley‐Liss, Inc.     

Membrane cholesterol depletion as a trigger of Nav1.9 channel‐mediated inflammatory pain

Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage‐gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol‐rich lipid rafts to cholesterol‐poor non‐raft regions of the membrane. Low‐cholesterol environment enhances voltage‐dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.     

Condensin minimizes topoisomerase II‐mediated entanglements of DNA in vivo

The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.     

Replication initiation and DNA topology: The twisted life of the origin

Genomic propagation in both prokaryotes and eukaryotes is tightly regulated at the level of initiation, ensuring that the genome is accurately replicated and equally segregated to the daughter cells. Even though replication origins and the proteins that bind onto them (initiator proteins) have diverged throughout the course of evolution, the mechanism of initiation has been conserved, consisting of origin recognition, multi‐protein complex assembly, helicase activation and loading of the replicative machinery. Recruitment of the multiprotein initiation complexes onto the replication origins is constrained by the dense packing of the DNA within the nucleus and unusual structures such as knots and supercoils. In this review, we focus on the DNA topological barriers that the multi‐protein complexes have to overcome in order to access the replication origins and how the topological state of the origins changes during origin firing. Recent advances in the available methodologies to study DNA topology and their clinical significance are also discussed. J. Cell. Biochem. 110: 35–43, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.     

Incorporation and uptake of thymidine and uridine by hela cells: Effect of DNA extracts prepared from normal human fibroblasts

Summary The incorporation and uptake of (³H) thymidine into HeLa cells markedly decreased in the presence of nuclear homogenates and DNA extracts that have been derived from normal diploid cell cultures. On the other hand, uridine uptake and incorporation were stimulated under the same conditions. The inhibition could be reversed immediately upon removal of the exogenous fraction from the culture medium. The inhibitory properties of the extracts are propagated by excreted cellular components as well as after DNAase treatment. The inhibitory factor is thermostable, resistant to pronase treatment, and seems to be related to nucleic acid. The material herein represents part of a dissertation presented by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Tel Aviv University, Israel. This work was partially supported by a grant from the Israel Ministry of Health.     

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.     

Cationic lipid-mediated gene transfer: Analysis of cellular uptake and nuclear import of plasmid DNA

Cationic lipids are widely used for gene transfer in vitro and show promise as vectors for in vivo gene therapy applications. However, there is limited understanding of the cellular mechanisms involved in nonviral gene transfer. We investigated two major steps that could be limiting barriers to cationic lipid-mediated gene transfer in vitro. We used a fluorescent plasmid to study the cellular uptake and the intracellular fate of lipoplexes during in vitro transfection of fibroblast cells and found that 100% of the cells take up lipoplexes. The intracellular staining observed with lipoplexes was clearly different from that obtained with endocytosed fluorescent dextran. This suggests that cells readily take up lipoplexes by a mechanism that could be different from endocytosis in our conditions. However, the escape of DNA from intracellular vesicles could be a major limiting barrier to gene transfer. Direct injection of plasmid DNA into the nucleus and cytoplasm of cells indicated that DNA traffic from the cytoplasm to the nucleus might be also an important limiting step.     

Molecular recognition of oligonucleotides and plasmid DNA by l‐methionine

The present study explores the effect of oligonucleotide composition on the mechanism of retention to l ‐methionine agarose support by chromatography and saturation transfer difference (STD)‐nuclear magnetic resonance (NMR) techniques. All chromatographic experiments were performed using 1.5 M (NH₄)₂SO₄. The binding profiles obtained by chromatography show that oligonucleotides with thymine had the highest retention time. In general, the larger homo‐oligonucleotides are more retained to the l ‐methionine agarose support. Moreover, the study with hetero‐oligonucleotides confirms that the presence of guanine reduces the retention on the l ‐methionine chromatographic support. These results are in accord with STD‐NMR experiments, which show that the strongest signals were observed for the methyl group of thymine, and no STD signals were observed for the guanosine protons. Finally, the retention behaviour of linear plasmid DNA (pDNA) with different sizes and base composition (2.7‐kbp pUC19, 6.05‐kbp pVAX1‐LacZ, 7.4‐kbp pVAX1‐LacZgag and 14‐kbp pcDNA‐based plasmid) was also evaluated by chromatography. The results indicate that the underlying mechanism of retention involves not only hydrophobic interactions but also other elementary interactions responsible for the biorecognition of pDNA molecules by l ‐methionine ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.     

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39°C) in a modified Tyrode's solution (TALP) containing (1) Chol-free liposomes (—Chol, 50 × 10⁶ sperm and 600 nmol phospholipid/ml); (2) liposomes containing 30 mol% Chol (+ Chol, 2 × 10⁸ sperm and 300 nmol total lipid/ml); or (3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with —Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 × 10⁶) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-μl droplets of TALP under medical fluid in an atmosphere of 5% CO₂ in air (3 h, 39°C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined. Percent penetration (%P) of HO (X ± SEM) for 30 and 40 μg of LPC/mg BSA was 59.4 ± 5.3 and 82.9 ± 5.4; 38.5 ± 5.6 and 52.3 ± 4.7; and 16.0 ± 4.6; and 23.2 ± 5.6 for —Chol, Control, and + Chol treatments, respectively (n = 3). %P of BO (X ± SEM) for 30, 35, and 40 μg of LPC/mg BSA was 43.3 ± 5.4, 70.7 ± 7.5, and 81.5 ± 5.1 for —Chol and 16.4 ± 6.9, 36.2 ± 6.9, and 44.2 ± 8.6 for Control treatments, respectively (n = 3). In a second set of experiments %P of BO (X ± SEM) was 63.6 ± 6.8, 31.8 ± 4.9, and 10.5 ± 3.4 for —Chol, Control, and +Chol treatments, respectively, when 40 μg LPC/mg BSA was added (n = 2). %P and the number of sperm per fertilized ovum were consistently higher for the —Chol treatment for both HO and BO (P < .01). These results demonstrate that Chol removal from bovine sperm facilitates penetration of ova in vitro suggesting a potential role in bovine sperm capacitation.     

Unusual sterolic mixture,and 24‐isopropylcholesterol,from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco‐2 cells

An unusual sterolic mixture (82.3% of 24‐isopropylated sterols) and its major component, 24‐isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT‐2 mRNA expression and increase the expression of ABCA1 mRNA in Caco‐2 cells. The decreases of cholesterol uptake and secretion induced by 24‐isopropylcholesterol alone were more than that of both the sterolic mixture and β‐sitosterol. These data add a new sterol, 24‐isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption. J. Cell. Biochem. 106: 659–665, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the interaction of a mono‐6‐thio‐β‐cyclodextrin‐capped CdTe quantum dots–methylene blue/methylene green system with herring sperm DNA using a spectroscopic approach

Novel, water‐soluble CdTe quantum dots (QDs) capped with β‐cyclodextrin (β‐CD) and ~ 4.0 nm in diameter were synthesized in aqueous solution, and characterized using transmission electron microscopy (TEM). A fluorescence‐sensing system based on the photoinduced electron transfer (PET) of (mono‐6‐thio‐β‐CD)–CdTe QDs was then designed to measure the interaction of phenothiazine dyes [methylene blue (MB) and methylene green (MG)] with herring sperm DNA (hsDNA). This fluorescence‐sensing system was based on a fluorescence “OFF–ON” mode. First, MB/MG adsorbed on the surface of (mono‐6‐thio‐β‐CD)–CdTe QDs effectively quenches the fluorescence of (mono‐6‐thio‐β‐CD)–CdTe QDs through PET. Then, addition of hsDNA restores the fluorescence intensity of (mono‐6‐thio‐β‐CD)–CdTe QDs, because hsDNA can bind with MB/MG and remove it from the as‐prepared (mono‐6‐thio‐β‐CD)–CdTe QDs. In addition, detailed reaction mechanisms of the (mono‐6‐thio‐β‐CD)–CdTe QDs–MB/MG–hsDNA solution system were studied using optical methods, by comparison with the TGA–CdTe QDs–MB/MG–hsDNA solution system. Copyright © 2014 John Wiley & Sons, Ltd.     

Integration and scaling of UV‐B radiation effects on plants: from DNA to leaf

A process‐based model integrating the effects of UV‐B radiation through epidermis, cellular DNA, and its consequences to the leaf expansion was developed from key parameters in the published literature. Enhanced UV‐B radiation‐induced DNA damage significantly delayed cell division, resulting in significant reductions in leaf growth and development. Ambient UV‐B radiation‐induced DNA damage significantly reduced the leaf growth of species with high relative epidermal absorbance at longer wavelengths and average/low pyrimidine cyclobutane dimers (CPD) photorepair rates. Leaf expansion was highly dependent on the number of CPD present in the DNA, as a result of UV‐B radiation dose, quantitative and qualitative absorptive properties of epidermal pigments, and repair mechanisms. Formation of pyrimidine‐pyrimidone (6‐4) photoproducts (6‐4PP) has no effect on the leaf expansion. Repair mechanisms could not solely prevent the UV‐B radiation interference with the cell division. Avoidance or effective shielding by increased or modified qualitative epidermal absorptance was required. Sustained increased UV‐B radiation levels are more detrimental than short, high doses of UV‐B radiation. The combination of low temperature and increased UV‐B radiation was more significant in the level of UV‐B radiation‐induced damage than UV‐B radiation alone. Slow‐growing leaves were more affected by increased UV‐B radiation than fast‐growing leaves.