Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.     

Effect of calcification on the mechanical stability of plaque based on a three-dimensional carotid bifurcation model

Background

This study characterizes the distribution and components of plaque structure by presenting a three-dimensional blood-vessel modelling with the aim of determining mechanical properties due to the effect of lipid core and calcification within a plaque. Numerical simulation has been used to answer how cap thickness and calcium distribution in lipids influence the biomechanical stress on the plaque.

Method

Modelling atherosclerotic plaque based on structural analysis confirms the rationale for plaque mechanical examination and the feasibility of our simulation model. Meaningful validation of predictions from modelled atherosclerotic plaque model typically requires examination of bona fide atherosclerotic lesions. To analyze a more accurate plaque rupture, fluid-structure interaction is applied to three-dimensional blood-vessel carotid bifurcation modelling. A patient-specific pressure variation is applied onto the plaque to influence its vulnerability.

Results

Modelling of the human atherosclerotic artery with varying degrees of lipid core elasticity, fibrous cap thickness and calcification gap, which is defined as the distance between the fibrous cap and calcification agglomerate, form the basis of our rupture analysis. Finite element analysis shows that the calcification gap should be conservatively smaller than its threshold to maintain plaque stability. The results add new mechanistic insights and methodologically sound data to investigate plaque rupture mechanics.

Conclusion

Structural analysis using a three-dimensional calcified model represents a more realistic simulation of late-stage atherosclerotic plaque. We also demonstrate that increases of calcium content that is coupled with a decrease in lipid core volume can stabilize plaque structurally.     

The solution structure of mucous glycoproteins: proton NMR studies of native and modified ovine submaxillary mucin

The solution structure of native and systematically modified ovine submaxillary mucin (OSM) has been probed by proton NMR spectroscopic methods. Most of the resonances in the spectra have been tentatively assigned to the peptide and O-linked disaccharide, alpha-N-acetylneuraminic acid 2----6 alpha-N-acetylgalactosamine protons. On the basis of the observed chemical shifts, spectral resolution, and behavior of the exchangeable protons it is concluded the mucin possesses internal segmental flexibility and exists in solution as a random coil peptide. No long-lived interresidue peptide or carbohydrate hydrogen bonds were detected. The removal of (i) the C8 and C9 carbons of the sialic acid residue, (ii) the entire sialic acid residue, and (iii) the complete disaccharide side chain resulted in no significant changes in peptide core conformation. A limited set of proton spin coupling constants and nuclear Overhauser enhancements has been obtained for the threonine glycopeptide side chains in native and modified mucin. The results are consistent with the previously reported conformations for the (1----6) linkage in oligosaccharides and the threonyl glycosidic linkage in glycopeptides. The OSM disaccharide may exist as a extended linear structure with rotational freedom about the GalNAc C5-C6 bond, while the threonine glycosidic linkage appears to be sterically constrained, although multiple conformations about the threonine C beta-O gamma bond may be allowed. The small chemical shift perturbations detected in the glycosylated threonine methyl protons and the GalNAc carbons upon removal of the terminal sialic acid residue are consistent with this model.     

High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

Background  

In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure.     

Amino group environments and metal binding properties of carbon-13 reductively methylated bovine alpha-lactalbumin

13C NMR spectroscopy has been used to study the amino group environments and metal binding properties of 13C reductively methylated bovine alpha-lactalbumin. Bovine alpha-lactalbumin is a Ca2+ metalloprotein containing 12 lysyl amino groups and a free amino terminus. All 13 amino groups can be 13C-dimethylated without altering Ca2+ binding or biological activity. pH titrations (chemical shift vs. pH) of this dimethylated protein reveal unique behavior for each of the 13 amino groups. The pKa values for the lysyl amino groups range from 9.1 to 10.8 while the pKa for the N-terminal amino group is 8.3. This relatively high pKa (by 1 pH unit) for the N-terminal supports its interaction in an ion pair as proposed by Warme et al. [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782]. Carbon-13 NMR studies further show that the removal of Ca2+ from the high-affinity binding site results in a conformational change, with the disruption of the N-terminal ion pair interaction (pKa decreased to 7.4). The study of Zn2+ binding to Ca2+-saturated protein suggests that Zn2+ binds initially at a low-affinity Ca2+ site while maintaining the N-terminal ion pair interaction. The further addition of Zn2+ leads to the disruption of this ion pair forming a presumed apoprotein-like conformation. Finally on the basis of the specific effects of added Mn2+ on the 13C NMR spectra of the methylated protein, a low-affinity divalent metal binding site is proposed about 7.5 A from the amino terminus.