Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Arachidonate incorporation into phospholipids in rat brain: A comparison between slice and membrane preparation

The incorporation of [³H]arachidonic acid ([³H]AA) into cerebral phospholipids was studied in slices and membranes of rat brain cortex. Effects of preincubation, different washing procedures with or without bovine serum albumin, or incubation in the presence of neurotransmitters were investigated. Over 60% of the phospholipid-bound [³H]arachidonic acid was recovered in phosphatidylinositol in both slice and membrane preparations. Preincubation or addition of bovine serum albumin resulted in slower incorporation of the isotope to membrane as well as to slice phospholipids, but removal of the added serum albumin enhanced the incorporation in the membrane preparation. Preincubation of slices in the presence of norepinephrine (1 mM) or serotonin (1 or 2.5 mM) resulted in a marked increase of [³H]arachidonate incorporation into phosphatidylinositol concomitant with decreased incorporation into other phosopholipids. Analysis of [³H]arachidonate incorporation as a function of time, in accordance with a compartmental model, indicated that observed differences in the accumulation of [³H]arachidonate in various phospholipids were probably due to different rates of acylation by arachidonyl-transferases with no appreciable changes in the rates of deacylation by enzymes of the phospholipase A type. Further, the pool of phospholipid-bound arachidonate which is readily available for deacylation-reacylation processes appears to be distributed differentially among various phospholipids with more than 50% of this pool located in phosphatidylinositol and only 0.75% of the phosphatidylinositol molecules appear to be active in deacylation-reacylation processes involving arachidonyl groups.     

On the analysis of fluorescence decay kinetics by the method of least-squares

Analysis of fluorescence decay kinetics aims at the determination of the analytic expression and the numerical values of the pertinent parameters which describe the decay process. In the well-known method of least-squares, one assumes a plausible functional form for the decay data and adjusts the values of the parameters until the statistically best fit is obtained between the data and the calculated decay function, i.e., until the sum of the weighted squares of the residuals is at a minimum. It is shown that proper weighting of the squares of the residuals may markedly improve the quality of the analysis. Such weighting requires information about the character of the experimental noise, which is often available, e.g., when the noise is due to counting error in photon-counting techniques. Furthermore, dramatic improvements in the accuracy of the analysis may often be achieved by use of auxiliary information available about the system studied. For example, the preexponents in a multiexponential fluorescence decay of a mixture of chromophores (such as tryptophan residues in a protein molecule) may sometimes be estimated independently; much higher accuracy can then be attained for the decay lifetimes by analysis of the decay kinetics. It is proposed that the shape of the autocorrelation function of the weighted residuals may serve as a convenient criterion for the quality of fit between the experimental data and the decay function obtained by analysis. The above conclusions were reached by analysis of computer-simulated experiments, and the usefulness of this approach is illustrated. The importance of stating the uncertainties in the estimated parameters inherent in the analysis of decay kinetics is stressed.     

Immunohistochemical studies with antibodies to myosins from the cytoplasm and membrane fraction of human blood platelets

Summary Antibodies were raised to myosins extracted from the cytoplasm and solubilized membranes of human blood platelets. Both antibodies had similar titers as shown by enzyme-immunoassay and bound to the same sites as shown by immunohistochemistry. They were specific for cytoplasmic myosins (e.g., in human white blood cells, platelets and fibroblasts and rat endothelial cells). They showed no crossreaction with human or rat smooth muscle.Abbreviations ATPase adenosine triphosphatase EC 3.6.1.3 - FITC fluorescein isothiocyanate     

A stochastic approach to risk management for prostate cancer patients on active surveillance

Screening for prostate cancer (PC) has led to more cancers being detected at early stages, where active surveillance (AS), a strategy that involves monitoring and intervention when the disease progresses, is an option. Physicians are seeking ways to measure progression of the disease such that AS is abandoned when appropriate. A blood test, prostate-specific antigen (PSA), and the concept of doubling time (PSADT) and PSA kinetics are being used as proxies of disease speed of progression. Studies using these proxies report conflicting results. These studies cast doubts on the current rules for stopping AS and recent research concludes that PSADT and PSA kinetics are unreliable triggers for intervention in an AS program. These findings are consistent with stochastic processes being analyzed as if they were “deterministic” (i.e., current models measure disease progression by PSA’s evolution assuming it to be deterministic). A model that best describes PSA evolution is a pre-requisite to the establishment of decision criteria for abandoning AS. This paper suggests modeling PSA evolutions and kinetics as stochastic processes. Consequently, triggers for stopping AS may be different than PSADT and can result in substantially different recommendations, which are likely to have significant impact on patients and the healthcare system.     

1,25-Dihydroxyvitamin D3 and agents that increase intracellular adenosine 3′, 5′-monophosphate synergistically inhibit fibroblast proliferation

Summary Agents that increase intracellular cAMP (cAMP elevating agents) and 1,25(OH)₂D₃ inhibit the proliferation of many cell types. We investigated the combined effect of 1,25(OH)₂D₃ and cAMP elevating agents on exponentially growing mouse 3T3 fibroblasts. The following cAMP elevating agents were used: theophylline and pentoxyfilline, which inhibit cAMP-dependent phosphodiesterase; prostaglandin E₂ which activates adenylate cyclase by a receptor-mediated mechanism; forskolin, which directly stimulates adenylate cyclase; and the cell permeable cAMP analogs 8-bromo cAMP and N⁶ benzoyl cAMP. 1,25(OH)₂D₃ and cAMP elevating agents were added to exponentially growing fibroblasts cultured in 96-well microtiter plates and cell number was monitored 3–7 d later. 1,25(OH)₂D₃ and the cAMP elevating agents as single agents inhibited the growth of the 3T3 cells. The combined treatment of the fibroblasts with 1,25(OH)₂D₃ and the cAMP elevating agents resulted in an antiproliferative effect that was more than additive. The synergistic interaction depended on the dose of 1,25(OH)₂D₃ and was apparent already at 10⁻⁸ M of the hormone. The specificity of the effect of 1,25(OH)₂D₃ was demonstrated by the finding that 24,25-dihydroxyvitamin D₃, a vitamin D metabolite with low affinity for the vitamin D receptor, did not affect the antiproliferative effect of cAMP elevating agents. From the synergistic interaction between 1,25(OH)₂D₃ and the cell permeable cAMP analogs, we infer that the site of interaction between the two signaling pathways is distal to the cAMP generating and degrading machinery.     

Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin‐like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C‐terminus of the target protein (protein‐ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP‐protein constructs have a lower specific activity than protein‐ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein‐ELP and ELP‐protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N‐terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C‐terminal end of the protein.     

Arousal increases the representational capacity of cortical tissue

Arousal patently transforms the faculties of complex organisms. Although typical changes in cortical activity such as seen in EEG and LFP measurements are associated with change in state of arousal, it remains unclear what in the constitution of such state dependent activity enables this profound enhancement of ability. We put forward the hypothesis that arousal modulates cortical activity by rendering it more fit to represent information. We argue that representational capacity is of a dual nature—it requires not only that cortical tissue generate complex activity (i.e. spatiotemporal neuronal events), but also a complex cortical activity space (which is comprised of such spatiotemporal events). We explain that the topological notion of complexity—homology—is the pertinent measure of the complexity of neuronal activity spaces, as homological structure indicates not only the degree to which underlying activity is inherently clustered but also registers the effective dimensionality of the configurations formed by such clusters. Changes of this sort in the structure of cortical activity spaces can serve as the basis of the enhanced capacity to make perceptual/behavioral distinctions brought about by arousal. To show the feasibility of these ideas, we analyzed voltage sensitive dye imaging (VSDI) data acquired from primate visual cortex in disparate states of arousal. Our results lend some support to the theory: first as arousal increased so did the complexity of activity (that is the complexity of VSDI movies). Moreover, the complexity of structure of activity space (that is VSDI movie space) as measured by persistent homology—a multi scale topological measure of complexity—increased with arousal as well.