N-hydroxymethylpentamethylmelamine,a major in vitro metabolite of hexamethylmelamine

N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.     

Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice

To identify markers of the earliest stage of atherosclerosis, endothelial dysfunction, we evaluated the gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in very young pre-atherosclerotic mice. Furthermore, the plasma levels of the soluble VCAM-1 and ICAM-1 were compared to the gene expression profiles. Gene expressions of LOX-1 and VCAM-1 were up-regulated in young apoE^−/− mice, and thus, it seems probable that these genes play a role in pre-atherosclerosis. Contrarily, the gene expression profile of ICAM-1 did not show any apparent differences between the groups, questioning the involvement of this molecule in the early development of atherosclerosis. Plasma levels of sVCAM-1 and sICAM-1 were similar in all mice and did not correlate with the vascular gene expression of the corresponding genes. It therefore seems likely that these circulating markers are not suited to detect early atherosclerosis.     

Prolonged mechanical ventilation induces cell cycle arrest in newborn rat lung

Rationale

The molecular mechanism(s) by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown.

Objective

To determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats.

Methods

Seven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg⁻¹).

Measurement and Main Results

Ventilation for 24 h (h) decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation) was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8–24 h of ventilation, while that of p27^Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57^Kip2, decreased that of p16^INK4a, while the levels of p21^Waf/Cip1 and p15^INK4b were unchanged. Increased p27^Kip1 expression coincided with reduced phosphorylation of p27^Kip1 at Thr¹⁵⁷, Thr¹⁸⁷ and Thr¹⁹⁸ (p<0.05), thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg⁻¹) and when fetal lung epithelial cells were subjected to a continuous (17% elongation) cyclic stretch.

Conclusion

This is the first demonstration that prolonged (24 h) of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G₁ and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27^Kip1, p57^Kip2) from the Kip family.     

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

Background

Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.

Methods

To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.

Results

The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.

Conclusions

Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.     

Nitrogen and carbon isotope variability in the green‐algal lichen Xanthoria parietina and their implications on mycobiont–photobiont interactions

Stable isotope patterns in lichens are known to vary largely, but effects of substrate on carbon and nitrogen stable isotope signatures of lichens were previously not investigated systematically. N and C contents and stable isotope (δ¹⁵N, δ¹³C) patterns have been measured in 92 lichen specimens of Xanthoria parietina from southern Bavaria growing on different substrates (bark and stone). Photobiont and mycobiont were isolated from selected populations and isotopically analyzed. Molecular investigations of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA) region have been conducted on a subset of the specimens of X. parietina. Phylogenetic analysis showed no correlation between the symbionts X. parietina and Trebouxia decolorans and the substrate, isotope composition, or geographic origin. Instead specimens grown on organic substrate significantly differ in isotope values from those on minerogenic substrate. This study documents that the lichens growing on bark use additional or different N sources than the lichens growing on stone. δ¹⁵N variation of X. parietina apparently is controlled predominantly by the mass fraction of the mycobiont and its nitrogen isotope composition. In contrast with mycobionts, photobionts of X. parietina are much more ¹⁵N‐depleted and show less isotopic variability than mycobionts, probably indicating a mycobiont‐independent nitrogen acquisition by uptake of atmospheric ammonia.     

Real‐Time Investigation of Intercalation and Structure Evolution in Printed Polymer:Fullerene Bulk Heterojunction Thin Films

The complex intermixing morphology is critical for the performance of the nanostructured polymer:fullerene bulk heterojunction (BHJ) solar cells. Here, time resolved in situ grazing incidence X‐ray diffraction and grazing incidence small angle X‐ray scattering are used to track the structure formation of BHJ thin films formed from the donor polymer poly(2,5‐bis(3‐hexadecylthiophen‐2‐yl)thieno[3,2‐b]thiophene) with different fullerene derivative acceptors. The formation of stable bimolecular crystals through the intercalation of fullerene molecules between the side chains of polymer crystallites is investigated. Such systems exhibit more efficient exciton dissociation but lower photo‐conductance and faster decay of charges. On the basis of the experimental observations, intercalation obviously takes place before or with the formation of the crystalline polymer domains. It results in more stable structures whose volume remains constant upon further drying. Three distinct periods of drying are observed and the formation of unidimensional fullerene channels along the π‐stacking direction of polymer crystallites is confirmed.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.     

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.