Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity

Aggregated and highly phosphorylated tau protein is a pathological hallmark of Alzheimer's disease (AD) and other tauopathies. We identified motifs of alternating polar and apolar amino acids within the microtubule-binding repeats of tau which were interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique instantly aggregating tau mutant containing longer stretches of polar/apolar amino acids without losing its microtubule-binding capacity. These modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser(422), and PHF-1) and conformational epitopes (MC-1 and Alz50) in cells. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. Thus, our mutant protein provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.     

The tumor suppressor RASSF1A is a novel effector of small G protein Rap1A

Rap1A is a small G protein implicated in a spectrum of biological processes such as cell proliferation, adhesion, differentiation, and embryogenesis. The downstream effectors through which Rap1A mediates its diverse effects are largely unknown. Here we show that Rap1A, but not the related small G proteins Rap2 or Ras, binds the tumor suppressor Ras association domain family 1A (RASSF1A) in a manner that is regulated by phosphorylation of RASSF1A. Interaction with Rap1A is shown to influence the effect of RASSF1A on microtubule behavior.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

Behavioral responses and bioefficacy of some aromatic amides against Aedes aegypti

A series of substituted aromatic amides by varying the chain length, substitution of methyl, methoxy, chloro, and fluoro groups at ortho-, meta-, and para-positions of the phenyl ring of N,N-diethyl-2-phenylacetamide were synthesized. Laboratory studies were carried out to observe the behavioral responses and repellent activity of these newly synthesized aromatic amides against Aedes aegypti (L.) mosquitoes. The deterrent activity of these synthetic amides against any vectors has not been reported previously. These aromatic amides were tested for their behavioral responses and compared with the well known insect repellents, namely, N,N-diethyl toluamide; N,N-diethyl phenylacetamide; and N,N-diethylbenzamide. Out of the 14 compounds synthesized, seven compounds were selected on the basis of those showing >75% of repellent response for the bioefficacy test on human volunteers. The potential use of lead compounds in personal protection management is discussed.     

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties

Some creatures living in extremely low temperatures can produce some special materials called “antifreeze proteins” (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach “AFP-Pred” for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.     

ATP synthase complex of Plasmodium falciparum: dimeric assembly in mitochondrial membranes and resistance to genetic disruption

The rotary nanomotor ATP synthase is a central player in the bioenergetics of most organisms. Yet the role of ATP synthase in malaria parasites has remained unclear, as blood stages of Plasmodium falciparum appear to derive ATP largely through glycolysis. Also, genes for essential subunits of the F(O) sector of the complex could not be detected in the parasite genomes. Here, we have used molecular genetic and immunological tools to investigate the localization, complex formation, and functional significance of predicted ATP synthase subunits in P. falciparum. We generated transgenic P. falciparum lines expressing seven epitope-tagged canonical ATP synthase subunits, revealing localization of all but one of the subunits to the mitochondrion. Blue native gel electrophoresis of P. falciparum mitochondrial membranes suggested the molecular mass of the ATP synthase complex to be greater than 1 million daltons. This size is consistent with the complex being assembled as a dimer in a manner similar to the complexes observed in other eukaryotic organisms. This observation also suggests the presence of previously unknown subunits in addition to the canonical subunits in P. falciparum ATP synthase complex. Our attempts to disrupt genes encoding β and γ subunits were unsuccessful, suggesting an essential role played by the ATP synthase complex in blood stages of P. falciparum. These studies suggest that, despite some unconventional features and its minimal contribution to ATP synthesis, P. falciparum ATP synthase is localized to the parasite mitochondrion, assembled as a large dimeric complex, and is likely essential for parasite survival.     

Oxidative cleavage of DNA by tridentate copper (II) complex

Copper (II) complex 1 having planar tridentate ligand, bzimpy, where bzimpy is 2,6-bis(benzimidazo-2-yl) pyridine was synthesized and characterized by UV-visible, FAB (fast atom bombardment) mass and infrared spectroscopy. From absorption titration data, the binding constant of Cu(II) with DNA was calculated to be (1.8+/-0.02)x10(4) M(-1). Thermal denaturation study of DNA with 1 revealed deltaT(m) of 5+/-0.5 degrees C. Viscosity measurement showed that complex binds with DNA through intercalative mode. Copper (II) complex induces cleavage in plasmid DNA in the presence of coreductants such as ascorbic acid or glutathione.