Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Predictors of post-stroke body temperature elevation

Background

Growing evidence indicates that elevated body temperature after stroke is associated with unfavorable outcome. The aim of the current study was to investigate which factors predict temperature elevation within 48 h of stroke onset. Specifically, we hypothesized that temperature elevation would be associated with stroke symptom severity and that hemorrhagic stroke would cause a more pronounced temperature increase compared to ischemic stroke.

Methods

The medical records of 400 stroke patients were retrospectively reviewed. Multiple linear regression analysis was used to determine which factors were associated with elevated body temperature.

Results

Several factors were significantly associated with peak body temperature (the highest recorded body temperature) within 48 h of stroke onset: stroke severity measured by the National Institutes of Health Stroke Scale (NIHSS) (regression coefficient; (RC) 0.022), female gender (RC 0.157), tympanic/non-rectal temperature reading (RC ?0.265), swallowing difficulties (RC 0.335), intubation (RC 0.470), antipyretic treatment (RC 0.563), and C-reactive protein?>?50 or signs of infection at admission (RC 0.298). Contrary to our expectations, patients with intracerebral hemorrhage did not have higher peak body temperatures than patients with ischemic stroke.

Conclusions

In conclusion, temperature elevation within the first 48 h of stroke onset is common, can be partially predicted using information at admission and is strongly associated with stroke severity. The strong association with stroke severity may, at least partly, explain the previously described association between post-stroke temperature elevation and unfavorable outcome.

     

Development of the deep‐sea viviparous quill worm Leptoecia vivipara (Hyalinoeciinae,Onuphidae, Annelida)

Development of Leptoecia vivipara, a brooding deep‐sea onuphid polychaete with a circum‐Antarctic distribution, was studied using light microscopy, scanning and transmission electron microscopy, and histology. All specimens examined were brooding viviparous females or juveniles; no male gametes were detected. Anterior segments of juvenile and adult worms bore paired compact ovaries with clusters of vitellogenic oocytes. In adults, the mid‐body region formed a chamber containing up to 12 offspring at different stages of development, from oocyte to 13 chaetigers. Mature oocytes freely floated in the coelomic fluid, while embryos and juveniles were enclosed in peritoneal envelopes. Chaetal replacement in juveniles and the morphology of the provisional maxillae are described. Leptoecia vivipara is argued to be a progenetic species with juvenile‐like external morphology and accelerated sexual maturation. These traits may have arisen as adaptations to epibenthic life in a high‐latitude deep‐sea environment affected by seasonal pulses of organic matter.     

Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to “Nostocoida limicola” and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.     

Miniaturized <Superscript>1</Superscript>H-NMR method for analyzing limited-quantity samples applied to a mouse model of Leigh disease

Introduction

The analysis of limited-quantity samples remains a challenge associated with mouse models, especially for multi-platform metabolomics studies. Although inherently insensitive, the highly specific characteristics of nuclear magnetic resonance (NMR) spectroscopy make it an advantageous platform for global metabolite profiling, particularly in mitochondrial disease research.

Objectives

Show method equivalency between a well-established standard operating protocol (SOP) and our novel miniaturized ¹H-NMR method.

Method

The miniaturized method was performed in a 2 mm NMR tube on a standard 500 MHz NMR spectrometer with a 5 mm triple-resonance inverse TXI probe at room temperature.

Results

Firstly, using synthetic urine spiked with low (50 µM), medium (250 µM) and high (500 µM) levels (n?=?10) of nine standards, both the SOP and miniaturized method were shown to have acceptable precision (CV?<?15%), relative accuracy (80–120%), and linearity (R²?>?0.95), except for taurine. Furthermore, statistical equivalence was shown using the two one-sided test. Secondly, pooled mouse quadriceps muscle extract was used to further confirm method equivalence (n?=?3), as well as explore the analytical dynamics of this novel approach by analyzing more-concentrated versions of samples (up to 10× concentration) to expand identification of metabolites qualitatively, with quantitative linearity. Lastly, we demonstrate the new technique’s application in a pilot metabolomics study using minute soleus muscle tissue from a mouse model of Leigh syndrome using Ndufs4 KO mice.

Conclusion

We demonstrate method equivalency, supporting our novel miniaturized ¹H-NMR method as a financially feasible alternative to cryoprobe technology—for limited-quantity biological samples in metabolomics studies that requires a volume one-tenth of the SOP.

     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.