Microfolding: conformational probability map for the alanine dipeptide in water from molecular dynamics simulations

A direct attack on the protein-folding problem has been initiated with the free energy perturbation methods of molecular dynamics. The complete conformational probability map for the alanine dipeptide is presented. This work uses the SPC model for the explicit hydration of the dipeptide. Free energy differences for the four observed minima (beta, alpha R, alpha L, C7ax) are given, and the free energy barriers between minima are outlined.     

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or Hin fI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.     

Tumor-peptide-pulsed dendritic cells isolated from spleen or cultured in vitro from bone marrow precursors can provide protection against tumor challenge

 Dendritic cells (DC) purified from murine spleen or generated in vitro from bone marrow precursors were compared for their respective abilities to stimulate T cell responses and provide tumor protection in vivo. In vitro incubation with synthetic tumor peptide conferred on both DC populations the ability to induce proliferation of tumor-peptide-specific T cells in vitro. Spleen DC were reproducibly about twofold more effective than bone-marrow-derived DC in this assay. Both DC populations could also induce cytotoxic activity in vivo. In vitro cytoxicity assays showed that, while cytotoxic activity induced by immunization with spleen DC was clearly peptide-specific, a high non-specific cytotoxic activity was consistently observed after immunization with bone-marrow-derived DC, whether peptide-pulsed or not. Regardless of such high non-specific activity in vitro, only tumor-peptide-pulsed DC could provide protection against subsequent inoculation of tumor cells. DC not pulsed with tumor peptide were ineffective. We conclude that DC isolated from spleen or generated in vitro from bone marrow precursors are suitable reagents for use in tumor vaccination studies. Received: 13 March 1997 / Accepted: 25 May 1997     

CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.     

Determinants of serum levels of surfactant proteins A and B and Clara cell protein CC16.

Increased leakage of surfactant proteins A and B (SP-A and SP-B) and Clara cell secretory protein (CC16) from the air spaces into the circulation occurs in a range of respiratory conditions. However, circulating levels depend not only on the rate of entry into the circulation, but also on the rate of clearance. In order to clarify the role of the kidney in the clearance of these proteins, serum levels were related to markers of glomerular filtration in 54 non-smoking patients with varying degrees of renal dysfunction, none of whom had respiratory disease or were receiving dialysis at the time of sampling. Serum SP-A was related to SP-B (r = 0.53, p < 0.001) and to CC16 (r = 0.33, p < 0.02). Similarly, SP-B was related to CC16 (r = 0.39, p < 0.004). Stepwise multiple linear regression analysis suggested that serum SP-A and SP-B are influenced by age (approximately 20 and approximately 25% of variance, respectively), whereas CC16 is determined by renal function and, to a lesser extent, by body weight (approximately 63% of variance in total). We conclude that CC16 is cleared from blood by the renal route, whereas SP-A and SP-B are not. Serum SP-A and SP-B are influenced by age, which we speculate reflects increased damage to the alveolocapillary barrier.     

Specific immuno capturing of the Staphylococcal superantigen toxic‐shock syndrome toxin‐1 in plasma

Toxic‐shock syndrome is primarily caused by the Toxic‐shock syndrome toxin 1 (TSST‐1), which is secreted by the Gram‐positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V_β‐specific T‐cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy‐chain antibodies (VHH) in the immuno capturing of TSST‐1 from plasma. Data is presented that the selected VHHs are highly specific for TSST‐1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST‐1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST‐1 more than 96% of TSST‐1 was depleted from pig plasma. These data pave the way to further explore application of high‐affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans. Biotechnol. Bioeng. 2009; 104: 143–151 © 2009 Wiley Periodicals, Inc.     

Cultured astrocytes derived from corpus callosum or cortical grey matter show distinct glutamate handling properties

While the astrocytic control of extracellular glutamate concentration at synaptic contacts is well characterized, little is known regarding the clearance of glutamate along axon tracts, even though local excitotoxic damage has been reported. Therefore, we have compared glutamate handling in astrocyte cultures derived from white matter (corpus callosum) and grey matter tissues (cortical structures). These populations of astrocytes showed clearly distinct phenotypes, adopting stellate or protoplasmic morphologies respectively. In addition, white matter astrocytes showed high densities of the intermediate filament proteins glial fibrillary acidic protein, vimentin and nestin. The glutamate–aspartate transporter and glutamate transporter‐1, as well as glutamine synthetase, were found to be expressed at higher levels in white matter compared with grey matter astrocytes. Consistent with this aspartate uptake capacity was three to fourfold higher in white matter cells, and the use of specific inhibitors revealed a substantial activity of glutamate transporter‐1, contrasting with grey matter cells where this transporter appeared poorly functional. In addition, expression of type 5 metabotropic glutamate receptors was considerably higher in white matter astrocytes where the agonist (S)‐3,5‐dihydroxyphenylglycine triggered a large release of intracellular calcium. Differences in these astrocyte cultures were also observed when exposed to experimental conditions that trigger glial activation. This study highlights typical features of cultured astrocytes derived from white matter tissues, which appear constitutively adapted to handle excitotoxic insults. Moreover, the expression and activity of the astroglial components involved in the control of glutamatergic transmission are reinforced when these cells are maintained under conditions mimicking a gliotic environment.