Conformational changes in the globin family during evolution

Summary Conformational restrictions imposed on the fixation of insertions and deletions by the three-dimensional structure of globins during evolution are analyzed. An evolutionary tree for the primary structure of 38 taxonomically distant globins was constructed by a computer method. Based on this tree, the calculated fixation frequency of point mutations was 50-fold higher than that established jointly for deletions and insertions, and the fixation frequency of deletions was more than three times that established for insertions. It was also found that deletions and insertions are predominantly fixed in the interhelical sections and at the ends of the -helices of the globin molecules. Conformational analysis of the packing of the -helices in the spatial structure of globins demonstrated that the fixation of deletions and insertions in the center of an -helix produces a marked distortion of their normal packing. The possible role of deletions and insertions in the evolution of protein families is discussed.     

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.     

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.     

The Impact of Tissue Morphology,Cross-Section and Turgor Pressure on the Mechanical Properties of the Leaf Petiole in Plants

This paper describes a study of petiole structural morphology in which tissue materials, cross-sectional geometry, layer-architecture and hydrostatic condition are variables that affect the overall structural properties of the organ. Philodendron melinonii is selected as a model species for characterizing the mechanical properties of the petiole. The shape of the petiole is modeled through the polar parameterization of the Lame's curves, i.e. Gielis formulation. A multiscale model of bending stiffness is proposed to capture the impact of changing the constituent tissues and the cross-sectional geometry. Stiffness and density of different tissues are used to plot the domain bounded by the limiting curve of the respective tissue material. Shape parameters and the respective tissue properties are used to generate structural efficiency maps displaying property domains within which fall all possible combinations of tissues that are shaped into a certain geometry during growth. The turgor pressure is also taken into account to show how the domain of the effective material properties changes with water content. Finite element analysis besides experimental data is used to validate the theoretical results. The maps may offer a source of inspiration for biomimetic design, as they help to gain insight into the efficiency of biological beams described by different tissues properties, geometry and turgidity.     

Interactions of recombinant prions with compounds of therapeutical significance

The transformation of the cellular prion protein (PrP(C)) into the infectious form (PrP(Sc)) is implicated in the invariably fatal transmissible spongiform encephalopathies. To identify a mechanism to prevent the undesired PrP(C)-->PrP(Sc) transformation, we investigated the interactions of recombinant prion proteins with a number of potential therapeutic agents which inhibit the PrP(Sc) formation, infectivity, and the accumulation of the misfolded form. We show that the prion aggregates formed in the presence of six compounds have no beta-structure, which is typical of the infectious form, and possess considerably higher alpha-helical content than the normal PrP(C). The investigated compounds stimulate the formation of alpha-helices and the destruction of beta-structure. They prevent the transformation of alpha-helical structure into beta-sheets. Probably, this is the reason for the resistance to PrP(C)-->PrP(Sc) transformation in the presence of these compounds. The results may be useful for the future therapy of neurodegenerative diseases.     

Annexin A5-conjugated quantum dots with a paramagnetic lipidic coating for the multimodal detection of apoptotic cells

Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim we developed a novel annexin A5-conjugated bimodal nanoparticle. The nanoparticle is composed of a quantum dot that is encapsulated in a paramagnetic micelle to enable its use both for optical imaging and MRI. Multiple recombinant human annexin A5 protein molecules were covalently coupled to the nanoparticle for targeting. In this study the specificity of the annexin A5-conjugated nanoparticles for apoptotic cells was demonstrated both with fluorescence microscopy and MRI, which confirms its potential for the detection of apoptosis with both imaging modalities in vivo.     

Computational model of excitable cell indicates ATP free energy dynamics in response to calcium oscillations are undampened by cytosolic ATP buffers

Mitochondria in excitable cells are recurrently exposed to pulsatile calcium gradients that activate cell function. Rapid calcium uptake by the mitochondria has previously been shown to cause uncoupling of oxidative phosphorylation. To test (i) if periodic nerve firing may cause oscillation of the cytosolic thermodynamic potential of ATP hydrolysis and (ii) if cytosolic adenylate (AK) and creatine kinase (CK) ATP buffering reactions dampen such oscillations, a lumped kinetic model of an excitable cell capturing major aspects of the physiology has been developed. Activation of ATP metabolism by low-frequency calcium pulses caused large oscillation of the cytosolic, but not mitochondrial ATP/ADP, ratio. This outcome was independent of net ATP synthesis or hydrolysis during mitochondrial calcium uptake. The AK/CK ATP buffering reactions dampened the amplitude and rate of cytosolic ATP/ADP changes on a timescale of seconds, but not milliseconds. These model predictions suggest that alternative sources of capacitance in neurons and striated muscles should be considered to protect ATP-free energy-driven cell functions.     

Cross-sectional geometry of the dentary in bats

Bats exhibit remarkable diversity in dietary habits, with species specializing on insects, fruit, nectar, vertebrates and blood. Studies of larger mammals have shown that structural differences in dentary cross-sectional properties exist among species with different diets. Unfortunately, few of these studies have considered the role of phylogeny in shaping these apparent form-function associations. Here we ask whether a relationship exists between diet and dentary structure in bats when phylogenetic history is factored into the analysis. To answer this question, we compared results from phylogenetic generalized least squares (PGLS) and traditional (nonphylogenetic) regression analyses of dentary cross-sectional shape in frugivorous, nectarivorous, and insectivorous bats (253 individuals representing 72 species). Cross-sectional moments of inertia of the dentary between M(1) and M(2) were computed from bone densitometry scans of skeletal specimens. Traditional regressions of cross-sectional parameters against dentary length detected significant departures from isometry among frugivores. In contrast, PGLS analyses indicated that cross-sectional variables for each dietary group scaled with isometry. Thus, the allometric patterns illuminated by traditional statistics are linked to the phylogenetic structure of the sample. Identical patterns of significant differences in slopes and intercepts between frugivores and nectarivores emerged from both traditional and PGLS analyses. As predicted, the cross-sectional shape of the dentary in frugivores is consistent with increased resistance to torsion and bending, while that of nectarivores suggested a less resistant dentary. Although traditional and PGLS analyses yielded some similar results, the phylogenetic structure of a sample can drive apparent patterns of scaling and should be considered in comparative functional analyses.