Detection of protein complex interactions via a Blue Native-PAGE retardation assay

We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M_R complex.     

Nucleosides with self-complementary hydrogen-bonding motifs: synthesis and base-pairing studies of two nucleosides containing the imidazo[4,5-d]pyridazine ring system

Synthesis and base-pairing studies of two 2'-deoxyribonucleosides, containing a common heterocyclic base, 7(4)-amino-5(6)H-imidazo[4,5-d]pyridazin-4(7)one (1 and 2), have been reported. The synthesis was accomplished by base-promoted deoxyribosylation of ethyl 5(4)-cyanoimidazole-4(5)-carboxylate (6), followed by ring-closure with hydrazine hydrate. The 1H NMR-based base-pair studies were conducted using DMF-d7 as a solvent by measuring changes in chemical shifts of the amino, hydrazide, imidazole H-2, and the sugar H-1' protons of the nucleosides with variations in concentrations and temperatures. Large downfield chemical shifts were observed for the NH, NH2, and to a lesser extent for the H-1' protons when the temperature was lowered from 25 to 0 degrees C, and then further down to -50 degrees C in 10 degree intervals. The observed experimental data are consistent with the results of molecular modeling studies. Nucleoside 2 exhibited low level antiviral activity against HIV-1 in CEM-SS cells with an IC50 of 89.2 microM. No cellular toxicity was observed at the highest concentration of the compound tested.     

Annual Rainfall and Seasonality Predict Pan-tropical Patterns of Liana Density and Basal Area

We test the hypotheses proposed by Gentry and Schnitzer that liana density and basal area in tropical forests vary negatively with mean annual precipitation (MAP) and positively with seasonality. Previous studies correlating liana abundance with these climatic variables have produced conflicting results, warranting a new analysis of drivers of liana abundance based on a different dataset. We compiled a pan-tropical dataset containing 28,953 lianas (≥2.5 cm diam.) from studies conducted at 13 Neotropical and 11 Paleotropical dry to wet lowland tropical forests. The ranges in MAP and dry season length (DSL) (number of months with mean rainfall <100 mm) represented by these datasets were 860–7250 mm/yr and 0–7 mo, respectively. Pan-tropically, liana density and basal area decreased significantly with increasing annual rainfall and increased with increasing DSL, supporting the hypotheses of Gentry and Schnitzer. Our results suggest that much of the variation in liana density and basal area in the tropics can be accounted for by the relatively simple metrics of MAP and DSL.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Discovery of matrix metalloproteases selective and activated peptide–doxorubicin prodrugs as anti-tumor agents

To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide–Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.     

Polarized light based scheme to monitor column performance in a continuous foam fractionation column

Background  

A polarized light scattering technique was used to monitor the performance of a continuously operated foam fractionation process. The S ₁₁ and S ₁₂ parameters, elements of the light scattering matrix, combined together (S ₁₁ +S ₁₂) have been correlated with the bubble size and liquid content for the case of a freely draining foam. The performance of a foam fractionation column is known to have a strong dependence on the bubble size distribution and liquid hold up in foam. In this study the enrichment is used as a metric, representative of foam properties and column performance, and correlated to the S ₁₁ +S ₁₂ parameter.     

A short survey on protein blocks

Protein structures are classically described in terms of secondary structures. Even if the regular secondary structures have relevant physical meaning, their recognition from atomic coordinates has some important limitations such as uncertainties in the assignment of boundaries of helical and β-strand regions. Further, on an average about 50% of all residues are assigned to an irregular state, i.e., the coil. Thus different research teams have focused on abstracting conformation of protein backbone in the localized short stretches. Using different geometric measures, local stretches in protein structures are clustered in a chosen number of states. A prototype representative of the local structures in each cluster is generally defined. These libraries of local structures prototypes are named as "structural alphabets". We have developed a structural alphabet, named Protein Blocks, not only to approximate the protein structure, but also to predict them from sequence. Since its development, we and other teams have explored numerous new research fields using this structural alphabet. We review here some of the most interesting applications.     

Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.