A simple rapid photometric estimation of the growth response of immobilized cells to fusaric acid and gamma radiation

A simple and rapid photometric method was developed in order to evaluate the growth responses of cells to various factors in vitro. Cells of Asparagus officinalis L. were mixed with autoclaved agar at 40°C and 90 l drops were dispensed into several Petri dishes. After the drops solidified, they were covered with a liquid growth medium and incubated on a gyratory shaker. Growth was measured every 2 or 3 days by two methods. In the first, the agar drop was placed under a stereomicroscope with substage illumination and the light passing through the embedded cells was measured by a light-meter. Growth, expressed by the increase in cell density, was inversely proportional to the intensity of transmitted light. In the second method, the agar drop was melted in a microwave oven and the packed cell volume was measured. The correlation between the two methods showed that the photometric method can be used to assess growth response of immobilized cells, during the first two weeks of culture. This method was used to evaluate growth responses to the toxin fusaric acid and to gamma radiation. The photometric method requires a small amount of inoculum, standard microscopic equipment and can be used to determine the effect of various factors on the growth of intact plant cells in vitro without disruption.Abbreviations FA fusaric acid - NAA naphthyl-1-acetic acid - CH casein hydrolysate - 2-i,P N⁶-(2-isopentyl) adenine - 2,4-D 2,4-dichloro-phenoxy acetic acid - PCV packed cell volume - gy grey     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The K_d of PYL5 for ABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.     

Two-layer passive/active anisotropic FSI models with fiber orientation: MRI-based patient-specific modeling of right ventricular response to pulmonary valve insertion surgery

A single-layer patient specific right/left ventricle patch (RV/LV/Patch) combination model with fluid-structure interactions (FSI) was introduced in our previous papers to evaluate and optimize human pulmonary valve replacement/insertion (PVR) surgical procedure and patch design. In this paper, an active anisotropic model with two-layer structure for ventricle wall and tissue fiber orientation was introduced to improve previous isotropic model for more accurate assessment of RV function and potential application in PVR surgery and patch design. A material-stiffening approach was used to model active heart contraction. The computational models were used to conduct "virtual (computational)" surgeries and test the hypothesis that a PVR surgical design with a smaller patch and more aggressive scar tissue trimming would lead to improved RV cardiac function recovery. Results from our models validated by pre-operation data indicated that the small patch design had 11% improvement in RV function as measured by RV ejection fraction, compared to the conventional patch. Maximum Stress-P1 value from the active anisotropic model was 121.2% higher than that from the passive isotropic model. Computational RV volume predictions agreed well with CMR-measured volume data (error < 2%).     

The origin of mitochondria in light of a fluid prokaryotic chromosome model

Biologists agree that the ancestor of mitochondria was an α-proteobacterium. But there is no consensus as to what constitutes an α-proteobacterial gene. Is it a gene found in all or several α-proteobacteria, or in only one? Here, we examine the proportion of α-proteobacterial genes in α-proteobacterial genomes by means of sequence comparisons. We find that each α-proteobacterium harbours a particular collection of genes and that, depending upon the lineage examined, between 97 and 33% are α-proteobacterial by the nearest-neighbour criterion. Our findings bear upon attempts to reconstruct the mitochondrial ancestor and upon inferences concerning the collection of genes that the mitochondrial ancestor possessed at the time that it became an endosymbiont.     

A full-genomic sequence-verified protein-coding gene collection for Francisella tularensis

The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications.