The role of PEI structure and size in the PEI/liposome-mediated synergism of gene transfection

Polyethylenimines (PEIs) and cationic liposomes are widely used for nonviral gene delivery. When PEIs have been used alone, the transfection efficiency has been higher for larger or linear than smaller or branched PEIs. We have reported previously that a combination of small PEIs and liposomes results in a potentiation of transfection efficiency in vitro. Here, the role of PEI size and structure in this synergism has been clarified further. Therefore, two structurally different high MW PEIs, i.e. the linear PEI22K and branched PEI25K, were studied in the SMC cells. We found that both linear PEI22K and branched PEI25K resulted in a similar synergism and comparable transfection efficiencies. However, the potentiation for larger PEIs found in the present study was weaker than that for smaller PEIs obtained in our previous studies. In conclusion, our present and previous results demonstrate that the increment of PEI/liposome-mediated gene transfection by different types of PEIs in vitro is a common attribute that is rather associated with their size than the structure. Interestingly, the effect of PEI size seems to be opposite when combined with liposome or given alone, i.e. the small PEIs are more effective when combined and less effective when alone than the larger ones.     

Pitfalls in the quantitative estimation of β-amyloid immunoreactivity in human brain tissue

 The duration of formic acid (FA) pretreatment clearly influences the extent of β-amyloid immunoreactivity in brain tissue and consequently also the results of quantitative analysis. All of the parameters studied (area fraction, density, and mean size of β-amyloid deposits) significantly increased with pretreatment of up to 6 h with β-amyloid antibody obtained from Dako. Longer exposure to FA only marginally increased the mean size of the single deposits, whereas the area fraction and the density of β-amyloid deposits slightly decreased. Optimal 6-h pretreatment (or even longer) did not reveal any β-amyloid aggregates in those cases where none was seen with shorter durations of FA pretreatment. Similar results were obtained with β-amyloid antibody 4G8 obtained from Senetek, whereas β-amyloid antibody 6E10 was shown to be less dependent upon FA pretreatment. In conclusion, we recommend that the FA pretreatment time should be studied and optimized for each antibody used and always be described when the quantitative analysis of β-amyloid load is reported. Accepted: 7 April 1998     

Peroxiredoxin II Expression and Its Association With Oxidative Stress and Cell Proliferation in Human Idiopathic Pulmonary Fibrosis

Oxidant burden has been suggested to be a contributor to the pathogenesis of idiopathic pulmonary fibrosis (IPF). The study focused on peroxiredoxin (Prx) II, an antioxidant that has been associated with platelet-derived growth factor (PDGF) signaling and consequent cell proliferation. Localization and expression of Prx II, PDGF receptors (PDGFRα, PDGFRβ), Ki67, and nitrotyrosine were assessed in control (n=10) and IPF/usual interstitial pneumonia (UIP) (n=10) lung biopsies by immunohistochemistry and morphometry. Prx II oxidation was determined by standard and non-reducing Western blots, two-dimensional gel electrophoresis, and mass spectrometry. Prx II localized in the IPF/UIP epithelium and alveolar macrophages. Prx II–positive area in the fibroblastic foci (FF) was smaller than in other parenchymal areas (p=0.03) or in the hyperplastic epithelium (p=0.01). There was no major Prx II oxidation in IPF/UIP compared with the normal lung. The FF showed only minor immunoreactivity to the PDGFRs; Ki67, a marker of cell proliferation; and nitrotyrosine, a marker of oxidative/nitrosative stress. The results suggest that Prx II oxidation does not relate to the pathogenesis of IPF/UIP and that Prx II, PDGFRs, and proliferating cells colocalize in the IPF/UIP lung. Unexpectedly, FF represented areas of low cell proliferation. (J Histochem Cytochem 56:951–959, 2008)     

A multi-metabolite analysis of serum by 1H NMR spectroscopy: early systemic signs of Alzheimer's disease

A three-molecular-window approach for ¹H NMR spectroscopy of serum is presented to obtain specific molecular data on lipoproteins, various low-molecular-weight metabolites, and individual lipid molecules together with their degree of (poly)(un)saturation. The multiple data were analysed with self-organising maps, illustrating the strength of the approach as a holistic metabonomics framework in solely data-driven metabolic phenotyping. We studied 180 serum samples of which 30% were related to mild cognitive impairment (MCI), a neuropsychological diagnosis with severely increased risk for Alzheimer’s disease (AD). The results underline the association between MCI and the metabolic syndrome (MetS). Additionally, the low relativeamount of ω-3 fatty acids appears more indicative of MCI than low serum ω-3 or polyunsaturated fatty acid concentration as such. The analyses also feature the role of elevated glycoproteins in the risk for AD, supporting the view that coexistence of inflammation and the MetS forms a high risk condition for cognitive decline.     

Assessing the current related heterogeneity and diversity patterns of benthic diatom communities in a turbid and a clear water river

Benthic diatoms were sampled in two rapids, in a turbid South-Finnish river (R. Keravanjoki, 22 FTU) and a clear water river in eastern Finland (R. Vaikkojoki, 4 FTU), to evaluate the diversity and spatial distribution patterns of diatom communities and especially their relationships to current velocity. In both rapids, epilithic diatoms were sampled in 15 sampling squares within three current velocity classes (10 cm s^–1, 40 cm s^–1 and 100 cm s^–1). The sampling squares had significantly different diatom communities in the three current velocity classes at both sampling sites, however, separation of the communities was much more pronounced in the clear water river (p < 0.001) than in the turbid river (p < 0.05). In the clear water river, communities at the highest velocity were highly different from those at the lower velocities. On the other hand, in the turbid river, communities were more similar at all velocities. Significant (p < 0.05) indicators for highest current velocity in the clear water R. Vaikkojoki were Fragilaria capucina var. gracilis Hustedt, F. capucina var. rumpens Lange-Bertalot and Meridion circulare Agardh. There were no significant indicators for high current velocity in the turbid R. Keravanjoki. Cocconeis placentula Ehr., Cymbella sinuata Gregory and Navicula lanceolata (Agardh) Ehr. were the three most abundant species in the highest velocity. This study showed that diatom community was highly specialized but low in diversity at the highest velocity in the clear water river. This pattern was not seen in the turbid R. Keravanjoki, indicating that the diatom community could withstand at the higher current velocities. In addition, the results show the importance of sampling in a variety of current regimes, particularly in clear water rivers, in order to properly assess the diatom diversity and community of a river section.     

High beta diversity of bacteria in the shallow terrestrial subsurface

While there have been a vast number of studies on bacterial alpha diversity in the shallow terrestrial subsurface, beta diversity - how the bacterial community composition changes with spatial distance - has received surprisingly limited attention. Here, bacterial beta diversity and its controlling factors are investigated by denaturing gradient gel electrophoresis and cloning of samples from a 700-cm-long sediment core, the lower half of which consisted of marine-originated sediments. According to canonical correspondence analysis with variation partitioning, contemporary environmental variables explain beta diversity in a greater proportion than depth. However, we also found that community similarity decayed significantly with spatial distance and the slopes of the distance-decay relationships are relatively high. The high beta diversity indicates that the bacterial distribution patterns are not only controlled by contemporary environments, but also related to historical events, that is, dispersal or depositional history. This is highlighted by the different beta diversity patterns among studied sediment layers. We thus conclude that the high beta diversity in the shallow terrestrial subsurface is a trade-off between historical events and environmental heterogeneity. Furthermore, we suggest that the high beta diversity of bacteria is likely to be recapitulated in other terrestrial sites because of the great frequency of high geochemical and/or historical variations along depth.     

N-Chloroacyl-6-O-triphenylmethylchitosans: useful intermediates for synthetic modifications of chitosan

An efficient synthetic route was developed for the mild chloroacylation of chitosan with different chloroacyl chlorides. Full N-chloroacylation was obtained with this procedure without any O-acylation, and products having lower degrees of substitution can also be produced. Organo-soluble 6-O-triphenylmethylchitosan was used as a starting material for the acylation reactions. The resulting N-chloroacyl-6-O-triphenylmethylchitosan intermediates were also organo-soluble and characterized by FT-IR. N-Methylpiperazine moieties were attached to make end products that were sufficiently soluble for characterization by NMR and also to prove that the present intermediates could be used for further modifications. The end products were fully characterized by 1H NMR, 13C NMR, and 2D 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. The degrees of substitution were determined by 1H NMR. Molecular weight determination by GPC-LS displayed a significant degradation of the polymer. The weight-average molar masses (M(w)) of the end products ranged from 29.6 to 49.4 kDa, when the M(w) of the starting material was 144.2 kDa.     

Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.