Similarity of fluorescence lifetime distributions for single tryptophan proteins in the random coil state.

The picosecond time-resolved fluorescence decay data of nine single-tryptophan (trp) proteins and two multi-trp proteins in their native and denatured states were analyzed by the maximum entropy method (MEM). In the denatured state (6 M guanidine hydrochloride) a majority of the single-trp proteins show bimodal (at 25 degrees C) and trimodal (at 85 degrees C) distributions with similar patterns and similar values for average lifetimes. In the native state of the proteins the lifetime distributions were bimodal or trimodal. These results (multimodal distributions) are contradictory to the unimodal Lorentzian distribution of lifetimes reported for some proteins in the native and denatured states. MEM analysis gives a unimodal distribution of lifetimes only when the signal-to-noise ratio is poor in the time-resolved fluorescence decay data. The unimodal distribution model is therefore not realistic for proteins in the native and denatured states. The fluorescence decay components of the bi- or trimodal distribution are associated with the rotamer structures of the indole moiety when the protein is in the random coil state.     

Mechanism of response of potential-sensitive dyes studied by time-resolved fluorescence

The mechanism of response of two potential-sensitive dyes, diOC₂(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC₂(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC₂(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye.     

MicroRNA-196a promotes renal cancer cell migration and invasion by targeting BRAM1 to regulate SMAD and MAPK signaling pathways

Rationale: MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play critical regulatory roles in various biological and pathological processes, including various cancers. Their function in renal cancer has not been fully elucidated. It has been reported that miR-196a can act as oncogenes or as tumor suppressors depending on their target genes. However, the molecular target for miR-196a and the underlying mechanism in miR-196a promoted cell migration and invasion in renal cancer is still not clear.Methods: The expression, survival and correlation between miR-196a and BRAM1 were investigated using TCGA analysis and validated by RT-PCR and western blot. To visualize the effect of Bram1 on tumor metastasis in vivo, NOD-SCID gamma (NSG) mice were intravenously injected with RCC4 cells (10⁶ cells/mouse) or RCC4 overexpressing Bram1. In addition, cell proliferation assays, migration and invasion assays were performed to examine the role of miR-196a in renal cells in vitro. Furthermore, immunoprecipitation was done to explore the binding targets of Bram1.Results: TCGA gene expression data from renal clear cell carcinoma patients showed a lower level of Bram1 expression in patients'' specimens compared to adjacent normal tissues. Moreover, Kaplan‑Meier survival data clearly show that high expression of Bram1correlates to poor prognosis in renal carcinoma patients. Our mouse metastasis model confirmed that Bram1 overexpression resulted in an inhibition in tumor metastasis. Target-prediction analysis and dual-luciferase reporter assay demonstrated that Bram1 is a direct target of miR-196a in renal cells. Further, our in vitro functional assays revealed that miR-196a promotes renal cell proliferation, migration, and invasion. Rescue of Bram1 expression reversed miR-196a-induced cell migration. MiR-196a promotes renal cancer cell migration by directly targeting Bram1 and inhibits Smad1/5/8 phosphorylation and MAPK pathways through BMPR1A and EGFR.Conclusions: Our findings thus provide a new mechanism on the oncogenic role of miR-196a and the tumor-suppressive role of Bram1 in renal cancer cells. Dysregulated miR-196a and Bram1 represent potential prognostic biomarkers and may have therapeutic applications in renal cancer.     

Photoluminescent coordination polymers of d metals with 4,4′-dipyridylsulfide (dps)

Reactions of AgClO₄, Zn(CH₃COO)₂ · H₂O and CuI with the ligand 4,4′-dipyridylsulfide (dps) in 1:1 ratio give rise to coordination polymers 1-3 and 5, the structures of which were characterized by X-ray crystallography. Polymers [Ag₂(dps)₂](ClO₄)₂ · MeCN (1) and [Ag₂(dps)₂(μ₂-MeCN)(MeCN)](ClO₄)₂ · MeCN · H₂O (2) are pseudo-supramolecular isomers, differing from each other in the coordination geometry of silver atom and the packing pattern. Both 1 and 2 are zigzag coordination polymers bridged by weak Ag?Ag, Ag?S or Ag?NC-CH₃ interactions to form double stranded coordination polymers. While [Zn(dps)(CH₃COO)₂] (3) is a zigzag single stranded coordination polymer, [Zn(dps)₂(H₂O)₂](ClO₄)₂ · H₂O (4) is an unusual mononuclear complex with a box-like structure. Interesting intermolecular hydrogen bonding present in the compounds 3 and 4 leads to 3D hydrogen-bonded network structure.Coordination polymer [Cu₂I₂(dps)₂] (5) is a non-interpenetrating (4,4) net. Photoluminescence properties of the compounds 1-5 have been examined in solid states at room temperature. These compounds have been found to exhibit yellow and blue photoluminescence.     

Orientation of beta-barrel proteins OmpA and FhuA in lipid membranes. Chain length dependence from infrared dichroism

The outer-membrane proteins OmpA and FhuA of Escherichia coli are monomeric beta-barrels of widely differing size. Polarized attenuated total reflection infrared spectroscopy has been used to determine the orientation of the beta-barrels in phosphatidylcholine host matrices of different lipid chain lengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples are used to deduce the strand tilt (beta = 46 degrees for OmpA and beta = 44.5 degrees for FhuA). These values are then used to deduce the order parameters, P(2)(cos alpha), of the beta-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the beta-barrels and their assembly in the membrane are discussed in terms of hydrophobic matching with the lipid chains.     

Life-threatening ventricular arrhythmia recognition by nonlinear descriptor

Background  

Ventricular tachycardia (VT) and ventricular fibrillation (VF) are ventricular cardiac arrhythmia that could be catastrophic and life threatening. Correct and timely detection of VT or VF can save lives.     

Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes

Functional assessments of cardiovascular fitness (CVF) are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT) is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO_2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT). Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J) to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO_2max, metabolic crossover) observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J) and genetic (cardiac isoform Casq2^-/-) models of cardiovascular dysfunction. Results showed that both tests reported VO_2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or the effects of novel therapeutics.     

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.

Methods

Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ³²P-GTP synthesised by NDK from GDP and γ³²P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound ³²P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni²⁺-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.

Results

NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.

Conclusion

Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.     

The role of skeletal‐muscle‐based thermogenic mechanisms in vertebrate endothermy

Thermogenesis is one of the most important homeostatic mechanisms that evolved during vertebrate evolution. Despite its importance for the survival of the organism, the mechanistic details behind various thermogenic processes remain incompletely understood. Although heat production from muscle has long been recognized as a thermogenic mechanism, whether muscle can produce heat independently of contraction remains controversial. Studies in birds and mammals suggest that skeletal muscle can be an important site of non‐shivering thermogenesis (NST) and can be recruited during cold adaptation, although unequivocal evidence is lacking. Much research on thermogenesis during the last two decades has been focused on brown adipose tissue (BAT). These studies clearly implicate BAT as an important site of NST in mammals, in particular in newborns and rodents. However, BAT is either absent, as in birds and pigs, or is only a minor component, as in adult large mammals including humans, bringing into question the BAT‐centric view of thermogenesis. This review focuses on the evolution and emergence of various thermogenic mechanisms in vertebrates from fish to man. A careful analysis of the existing data reveals that muscle was the earliest facultative thermogenic organ to emerge in vertebrates, long before the appearance of BAT in eutherian mammals. Additionally, these studies suggest that muscle‐based thermogenesis is the dominant mechanism of heat production in many species including birds, marsupials, and certain mammals where BAT‐mediated thermogenesis is absent or limited. We discuss the relevance of our recent findings showing that uncoupling of sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) by sarcolipin (SLN), resulting in futile cycling and increased heat production, could be the basis for NST in skeletal muscle. The overall goal of this review is to highlight the role of skeletal muscle as a thermogenic organ and provide a balanced view of thermogenesis in vertebrates.