RESUMEN
This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).
Asunto(s)
Guanidina , Acrilamidas , Guanidina/química , Guanidina/farmacología , Células HeLa , Humanos , Indoles , Plásmidos , TransfecciónRESUMEN
Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve elementary steps of the neuronal fusion process. However, in previous studies, we monitored only lipid mixing between fusing large unilamellar vesicles and PSMs and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into large unilamellar vesicles containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on gold-coated porous silicon substrates. By dual-color spinning disk fluorescence microscopy with a time resolution of â¼20 ms, we could unambiguously distinguish between bursting vesicles, which was only rarely observed (<0.01%), and fusion pore formation. From the time-resolved dual-color fluorescence time traces, we were able to identify different fusion pathways, including remaining three-dimensional postfusion structures with released content and transient openings and closings of the fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation after the merger of the two lipid membranes occurs almost simultaneously.
Asunto(s)
Fusión de Membrana , Proteínas SNARE , Microscopía Fluorescente , Liposomas Unilamelares , Proteína 2 de Membrana Asociada a VesículasRESUMEN
Environmental triggers and genetic factors are supposed to lead to complex gene expression changes in psoriasis and interact in the manifestation of the disease. The histamine H4 receptor (HRH4) is functionally expressed on Th17 cells and plasmacytoid dendritic cells (pDCs) which play a prominent role in the pathogenesis of psoriasis. On pDCs a higher basal expression level of the HRH4 in psoriasis patients compared to healthy controls has been detected. The functional relationship between predisposing genetic variations in the HRH4 gene and psoriasis is yet not known. The aim of the study was to evaluate a possible association between single nucleotide polymorphisms (SNPs) in the HRH4 gene primarily in the promotor region and incidence, severity as well as special clinical features (nail involvement, arthritis, palmoplantar location) of psoriasis. For this approach genomic DNA from 206 patients with psoriasis and 213 healthy controls of Caucasian origin was extracted and three SNPs in the promotor region and one SNP located in an intron of the HRH4 gene were analysed by PCR and pyrophosphate DNA-sequencing. The genotype distributions and allele frequencies between the different groups were compared by chi-square test. The analysis of association between HRH4 polymorphisms and psoriasis was assessed by odds ratio with 95% confidence interval. The genotype distributions and allele frequencies of the four SNPs in the HRH4 gene did not show obvious differences between the whole group of psoriasis patients and healthy controls. However, there were differences by trend in subgroup analysis: The mutant genotypes (A/G) of rs17203314 and (G/A) of rs615283 were more frequent in patients with severe psoriasis PASI≥30 (34.8% and 34.8%) when compared to the control groups (23.5% and 27.2%), respectively. The mutant G/A genotype of rs615283 was significantly more frequent in patients with moderate-to-severe psoriasis PASI≥10 when compared to mild psoriasis PASI<10 (33.3% vs 21.7%, p=0.022). For rs524149 and rs17797945 the wildtype CC genotype was more frequent by trend in moderately-to-severely affected patients with PASI≥10 (85.2% and 63.0%) when compared to the group with mild psoriasis PASI<10 (77.0% and 49.4%), respectively. Furthermore, a significant association of rs615283 with psoriasis palmoplantaris was detected. In conclusion our study suggests that genetic variations within the HRH4 gene might be associated with special clinical features of psoriasis. Further studies are needed in larger study populations to confirm the reported associations and investigate the functional relevance of the identified SNPs.