Engineering antibodies and proteins for molecular in vivo imaging

The rapid and ongoing discovery of new disease related biomarkers leads to a dramatic paradigm change in human healthcare and constitutes the basis for a truly personalized medicine. Molecular imaging enables early detection and classification of human diseases and provides valuable data for optimized, target-oriented therapies. By now, the biochemical and physiological properties of antibody derivatives or alternative protein scaffolds can be engineered for the detection of a wide range of target structures. The successful application of these reagents in animals, xenograft models and cells in preclinical research clearly demonstrate their utility for molecular imaging. Despite these promising perspectives, only a few antibodies and recombinant proteins are used yet for molecular imaging in human medicine. Especially the high safety demands and the need to eliminate off target effects in humans require extensive research and development efforts.     

Scission of the lactyl ether bond of N-acetylmuramic acid by Escherichia coli "etherase"

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Spectroscopy on individual light-harvesting 1 complexes of Rhodopseudomonas acidophila

In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.     

Cloning and characterization of the SIL promoter

The SIL gene undergoes a site-specific rearrangement with the SCL gene in 25% of patients with T cell acute lymphoblastic leukemia (ALL). The functional result of this rearrangement is that the SIL regulatory elements aberrantly drive expression of the SCL gene. We have cloned and sequenced the human SIL promoter, cloned a murine homolog, found the sequence to be highly conserved, and defined a minimal promoter region. Both the cloned murine and human sequences were found to be highly active in either human or murine cells. SCL mRNA, driven by a cloned SIL promoter, could be downregulated by DMSO in stably transfected F4-6 murine erythroleukemia cells. The SIL promoter was found to be partially unmethylated in proliferating tissues, in which it is highly expressed, and more highly methylated in post-mitotic tissues, in which SIL is not expressed. The isolation of the SIL promoter provides an important tool for the study of both the SIL gene expression as well as the role of the SIL promoter in leukemogenesis.     

Acetaminophen inhibits cytochrome c redox cycling induced lipid peroxidation

Recent evidence indicates that site-specific crosstalk between O-GlcNAcylation and phosphorylation and the O-GlcNAcylation of kinases play an important role in regulating cell signaling. However, relatively few kinases have been analyzed for O-GlcNAcylation. Here, we identify additional kinases that are substrates for O-GlcNAcylation using an in vitro OGT assay on a functional kinase array. Forty-two kinases were O-GlcNAcylated in vitro, representing 39% of the kinases on the array. In addition, we confirmed the in vivo O-GlcNAcylation of three identified kinases. Our results suggest that O-GlcNAcylation may directly regulate a substantial number of kinases and illustrates the increasingly complex relationship between O-GlcNAcylation and phosphorylation in cellular signaling.     

Pericardial fat and myocardial perfusion in asymptomatic adults from the Multi-Ethnic Study of Atherosclerosis

Background

Pericardial fat has adverse effects on the surrounding vasculature. Previous studies suggest that pericardial fat may contribute to myocardial ischemia in symptomatic individuals. However, it is unknown if pericardial fat has similar effects in asymptomatic individuals.

Methods

We determined the association between pericardial fat and myocardial blood flow (MBF) in 214 adults with no prior history of cardiovascular disease from the Minnesota field center of the Multi-Ethnic Study of Atherosclerosis (43% female, 56% Caucasian, 44% Hispanic). Pericardial fat volume was measured by computed tomography. MBF was measured by MRI at rest and during adenosine-induced hyperemia. Myocardial perfusion reserve (PR) was calculated as the ratio of hyperemic to resting MBF.

Results

Gender-stratified analyses revealed significant differences between men and women including less pericardial fat (71.9±31.3 vs. 105.2±57.5 cm³, p<0.0001) and higher resting MBF (1.12±0.23 vs. 0.93±0.19 ml/min/g, p<0.0001), hyperemic MBF (3.49±0.76 vs. 2.65±0.72 ml/min/g, p<0.0001), and PR (3.19±0.78 vs. 2.93±0.89, p = 0.03) in women. Correlations between pericardial fat and clinical and hemodynamic variables were stronger in women. In women only (p = 0.01 for gender interaction) higher pericardial fat was associated with higher resting MBF (p = 0.008). However, this association was attenuated after accounting for body mass index or rate-pressure product. There were no significant associations between pericardial fat and hyperemic MBF or PR after multivariate adjustment in either gender. In logistic regression analyses there was also no association between impaired coronary vasoreactivity, defined as having a PR <2.5, and pericardial fat in men (OR, 1.18; 95% CI, 0.82–1.70) or women (OR, 1.11; 95% CI, 0.68–1.82).

Conclusions

Our data fail to support an independent association between pericardial fat and myocardial perfusion in adults without symptomatic cardiovascular disease. Nevertheless, these findings highlight potentially important differences between asymptomatic and symptomatic individuals with respect to the underlying subclinical disease burden.     

Importance of feature selection in decision-tree and artificial-neural-network ecological applications. Alburnus alburnus alborella: A practical example

Recent advances in computing technology have increased interest in applying data mining to ecology. Machine learning is one of the methods used in most of these data mining applications. As is well known, approximately 80% of the resources in most data mining applications are devoted to cleaning and preprocessing the data. However, there are few studies on preprocessing the ecological data used as the input in these data mining systems. In this study, we use four different feature selection methods (χ², Information Gain, Gain Ratio, and Symmetrical Uncertainty) and evaluate their effectiveness in preprocessing the input data to be used for inducing artificial neural networks (ANNs) and decision trees (DTs). The presence/absence of fish is the data item used to illustrate our models. Feature selection is fundamental in order to increase the performances of the models obtained. Accuracy of classification improves when a small set of optimally selected features is used. DTs and ANNs are very useful tools when applied to modeling presence/absence of Alburnus alburnus alborella. ANNs generally performed better than DT models.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.