Polyethyleneimine-mediated gene delivery into human adipose derived stem cells.

In this study, we examined the use of polyethyleneimine (PEI) as a carrier for gene delivery in human adipose tissue-derived stem cells (hADSCs). These multipotent cells can form bone, cartilage, adipose, and other connective tissues. In primary culture, hADSCs are fibroblastic in appearance in primary culture, and they show a high rate of proliferation for at least five passages. Immunophenotyping showed that these cells are positive for the mesenchymal stem cell markers CD29 and CD44 but negative for the hematopoietic cell surface markers CD34, CD45, and c-kit. PEI and Lipofectamine were compared as gene carriers for hADSCs. DNA completely bound PEI at a negative-to-positive (N/P) charge ratio of 4. The PEI-DNA complexes were spherical with smooth surfaces. As the proportion of PEI was increased, the size of the PEI-DNA complexes decreased from 990 to 130nm, the positive surface charge decreased, and the cytotoxicity increased. Flow cytometry revealed that the transfection efficiency using PEI at N/P charge ratios of 4 and 8 was higher than that of Lipofectamine. The highest transfection efficiency (19%) was obtained at an N/P charge ratio of 8. After transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of hADSCs at 4h 30m and localize over cytoplasm from 9h 30m. In conclusion, PEI acts as an effective gene carrier for hADSCs.

Association of Some Vitamins and Minerals with Periodontitis in a Nationally Representative Sample of Korean Young Adults.

The purpose of this study was to investigate whether the intakes of some kinds of vitamins and minerals are associated with periodontitis in a nationally representative sample of young adults. This study comprised 2049 young adults aged 19-39 years who took both periodontal examination and nutrition survey. The vitamin and mineral intakes were calculated from dietary intake data gained by complete one-day 24-h recall interviews, and the intake levels for each nutrient were classified by the Recommended Nutrient Intake (RNI) in Dietary Reference Intakes for Koreans and median values. Periodontitis was assessed using Community Periodontal Index (CPI). Multivariate logistic regression analyses were performed in a whole sample and subgroups with the strata of gender or smoking, following a complex sampling design. In analyses according to RNI, a lower intake of niacin was significantly associated with periodontitis in young adults (odd ratio [OR] 1.47, 95% confidential interval [CI] 1.09-2.00) and in its subgroup of women (OR 1.70; 95% CI 1.10-2.64) and current non-smokers (OR 1.75; 95% CI 1.22-2.51). Whereas, in analyses according to median intake values, there were significant associations of periodontitis with a lower intake of niacin in women (OR 1.58; 95% CI 1.02-2.46) and current non-smokers (OR 1.50; 95% CI 1.01-2.22), with lower intake of vitamin C in women (OR 1.66; 95% CI 1.04-2.64) and in current non-smokers (OR 1.49; 95% CI 1.04-2.14), with lower intake of iron in women (OR 1.85; 95% CI 1.11-3.07), and with lower intake of vitamin A marginally in women (OR 1.56; 95% CI 1.00-2.44). In young adults, periodonitis is significantly associated with the lower intakes of niacin, vitamin C, and iron, especially in women and current non-smokers.

Synthesis and characterization of tri(ethylene oxide)-attached poly(amidoamine) dendrimer layers on gold.

This paper describes the synthesis of a tri(ethylene oxide)-attached fourth-generation poly(amidoamine) dendrimer (EO3-dendrimer) and the characterization of its layers on gold. NMR analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that about 61 amine groups of a G4 PAMAM dendrimer were covalently conjugated with tri(ethylene oxide) units, accounting for a 95% modification level. Layers of the EO3-dendrimer were formed on gold, and the resulting surface was characterized by infrared reflection absorption spectroscopy, ellipsometry, and contact angle goniometry. The EO3-dendrimer resulted in more hydrophilic and less compact layers with no substantial deformation of the molecule during layer formation by virtue of the EO3 units, compared to a PAMAM dendrimer. Interestingly, the specific binding of avidin to the biotinylated layers of the EO3-dendrimer approached a surface density of 5.2 +/- 0.2 ngmm-2, showing about 92% of full surface coverage. The layers of the EO3-dendrimer were found to be more resistant to nonspecific adsorption of proteins than PAMAM dendrimer layers when bovine serum albumin and serum proteins were tested.

Polyethyleneimine-mediated gene delivery into rat pheochromocytoma PC-12 cells.

In this study, we examined the use of polyethyleneimine (PEI) as a non-viral gene carrier and lipofectamine(trade mark) 2000 as control for rat pheochromocytoma PC-12 cells. The complex formation of PEI and DNA or lipofectamine and DNA was characterized by gel electrophoresis and measurement of particle size and surface charge. A gradual increase in surface charge (from 0.7 to 43 mV) and a gradual decrease in particle size (from 900 to 130 nm) was observed in the PEI-DNA complex with higher PEI concentrations. The cytotoxicity of PC-12 cells for lipofectamine-DNA complex was similar to PEI-DNA complex at N:P charge ratios of 4 and 8. Transfection efficiency was 14% for lipofectamine and 15% for PEI. At low N:P ratio, DNA condenses poorly, so the particle size tends to be large and polydispersed, resulting in poor transfection efficiency. Meanwhile, a high N:P ratio results in high transfection efficiency and cytotoxicity. Transfected PC-12 cells showed the generation of neurites from transfected PC-12 cells in the presence of NGF, indicating the differentiation of PC-12 cells. NGF-differentiated PC-12 cells were transfected by PEI-DNA complex of N:P charge ratio 8. From real-time imaging for transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of PC-12 cells at 5 h and localized in the cytoplasm from 15 h. Our study demonstrates that PEI or lipofectamine may be applied as an effective gene carrier for PC-12 cells.

A Family Harboring CMT1A Duplication and HNPP Deletion.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion.

Charcot-marie-tooth disease: seventeen causative genes.

Charcot-Marie-Tooth disease (CMT) is the most common form of inherited motor and sensory neuropathy. Moreover, CMT is a genetically heterogeneous disorder of the peripheral nervous system, with many genes identified as CMT-causative. CMT has two usual classifications: type 1, the demyelinating form (CMT1); and type 2, the axonal form (CMT2). In addition, patients are classified as CMTX if they have an X-linked inheritance pattern and CMT4 if the inheritance pattern is autosomal recessive. A large amount of new information on the genetic causes of CMT has become available, and mutations causing it have been associated with more than 17 different genes and 25 chromosomal loci. Advances in our understanding of the molecular basis of CMT have revealed an enormous diversity in genetic mechanisms, despite a clinical entity that is relatively uniform in presentation. In addition, recent encouraging studies - shown in CMT1A animal models - concerning the therapeutic effects of certain chemicals have been published; these suggest potential therapies for the most common form of CMT, CMT1A. This review focuses on the inherited motor and sensory neuropathy subgroup for which there has been an explosion of new molecular genetic information over the past decade.

Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion.

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.