Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic Iiposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining Iipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Lipoplex size determines lipofection efficiency with or without serum

In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.     

Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.     

Estimation of mental effort in learning visual search by measuring pupil response

Perceptual learning refers to the improvement of perceptual sensitivity and performance with training. In this study, we examined whether learning is accompanied by a release from mental effort on the task, leading to automatization of the learned task. For this purpose, we had subjects conduct a visual search for a target, defined by a combination of orientation and spatial frequency, while we monitored their pupil size. It is well known that pupil size reflects the strength of mental effort invested in a task. We found that pupil size increased rapidly as the learning proceeded in the early phase of training and decreased at the later phase to a level half of its maximum value. This result does not support the simple automatization hypothesis. Instead, it suggests that the mental effort and behavioral performance reflect different aspects of perceptual learning. Further, mental effort would be continued to be invested to maintain good performance at a later stage of training.     

Eicosapentaenoic acid attenuates statin-induced ER stress and toxicity in myoblast

T cell activation requires both antigen specific and co-stimulatory signals that include the interaction of CD28 with its ligands CD80 and CD86. These signals are delivered by antigen presenting cells (APC) in the context of the immunological synapse (IS). Reorganization of the cytoskeleton is required for the formation and maintenance of the IS. Our results show that a highly conserved polylysine motif in CD86 cytoplasmic tail, herein referred to as the K4 motif, is responsible for the constitutive association of CD86 to the cytoskeleton in primary human APC as well as in a murine APC model. This motif is not involved in initial APC:T cell conjugate formation but mutation of the K4 motif affects CD86 reorientation at the IS. Importantly, APCs expressing CD86 with mutated K4 motif are severely compromised in their capacity to trigger complete T cell activation upon peptide presentation as measured by IL-2 secretion. Altogether, our results reveal the critical importance of the cytoskeleton-dependent CD86 polarization to the IS and more specifically the K4 motif for effective co-signaling.     

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.     

Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB,Novel Targets of Eicosapentaenoic Acid

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.     

Distinct regulation of plasma LDL cholesterol by eicosapentaenoic acid and docosahexaenoic acid in high fat diet-fed hamsters: Participation of cholesterol ester transfer protein and LDL receptor

Despite established anti-atherogenic action, previous reports have shown that fish oils or n-3 poly-unsaturated fatty acid (PUFA) increase plasma LDL-C in animals and humans. However, which component of n-3 PUFAs and what mechanisms contribute to this increase are unclear. We investigated the effects of the major components of n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on plasma LDL-C in high fat diet-fed hamsters. While LDL-C increased significantly with n-3 PUFA oil and DHA, EPA had no effect on LDL-C. Interestingly, a positive correlation was found between plasma cholesterol ester transfer protein (CETP) activity and LDL-C. Only DHA increased plasma CETP activity and significantly decreased LDL receptor expression in the liver. Our data suggest that DHA, not EPA, is a major factor in the LDL-C increasing effect of n-3 PUFA oil. These differential effects on LDL-C may arise from differences in plasma CETP activity and LDL receptor expression.     

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.