The biophysical aspects of reconstructing a single cell by the methods of cell engineering

The problem of the low efficiency of mammalian cloning is discussed with emphasis on the need of expert assessment of every step in single cell reconstruction, beginning with microsurgical manipulations. Experimental proof is provided for the impairment of cell integrity upon its fixation for microsurgery by the negative pressure in a conventional holding pipette. The ensuing leakage of the cell contents is shown to depend on the value of negative pressure, the duration of holding, and the size of the holder orifice. An alternative method of cell fixation is proposed, taking advantage of the capillary forces in the holding micropipette. This reduces the holding effort by two orders of magnitude and raises the cell survival upon microsurgery at least to 92%. To alleviate cell damage by instrumental invasion, a new technique is proposed for making micropipettes. Another novel method is offered for pipette filling with viscous liquid such as DNA solution, which allows continuous injection of more than 1000 cells.     

Water permeability of rat liver mitochondria: A biophysical study

The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.     

Water permeability of rat liver mitochondria: A biophysical study

The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.     

Two methods for using period length to study rhythmic phenomena

Summary We have developed two distinct methods of biological rhythm analysis. The procedures are based on existing techniques for analysis of time series, Enright's periodogram and autocorrelation, and both of the new methods use the parameter, period length (), for defining oscillatory phenomena. We empirically evaluated the two types of analyses using real biological data from circadian rhythm studies in salamanders and sparrows.The first method permits us to make a statistical comparison of period lengths between groups of animals in given treatments. This method is useful for data where the signal-to-noise ratio of the suspected rhythm is very low; and the method is not adequate for making a definitive judgment from single animals. It can best be applied to the question of whether a signal is entraining a rhythm or not and to questions of group differences in period length.With the second method, we determined period length versus time. Using this procedure, we took into consideration the observation that the period length of many biological oscillations changes with time. The method is applicable to records from individual animals, and it can be used to compare treatment effects in individual animals. The technique can also be used to answer the common question of whether periodicityper se exists within a defined range in a time series.We thank Dr, T. J. Crovello, Mr. Kilian, Mr. B. Bailey, Mr. E. Kluth, Mr. G. Wyche, and the Notre Dame Computer Center. Support was provided by postdoctoral fellowship (l F02-HD-52858) from NIH to S. Binkley; grants to K. Adler (NSF GB-30547 and NIH FR-07 033-05); and NSF postdoctoral fellowship (GU-2058) and an Indiana Academy of Science grant to D. Taylor. Sparrow data used in this report were gathered while S. Binkley was a graduate student at the U. of Texas in Austin (NIH traineeship 5T01 GM-00836-08) and with funds from an NIH program project grant (HD-03803-02) to M. Menaker.     

Application of multivariate resolution methods to the study of biochemical and biophysical processes

Multivariate resolution methods make up a set of mathematical tools that may be applied to the analysis and interpretation of spectroscopic data recorded when monitoring a physical or chemical process with multichannel detectors. The goal of resolution methods is the recovery of chemical and/or physical information from the experimental data. Such data include, for example, the number of intermediates present in a reaction, the rate or equilibrium constants, and the spectra for each one of those intermediates. Multivariate resolution methods have been shown to be useful for the study of biophysical and biochemical processes such as folding/unfolding of proteins or nucleic acids. The present article reviews the most frequently used resolution methods, the limitations on their use, and their latest applications in protein and nucleic acid research.     

Constants underlying frequency changes in biological rhythmic movements

When an animal increases or decreases the frequency of its limb motions, how should the transformation in timing be characterized? It has been hypothesized that the transformation is adiabatic, even though the biological conditions are nonconservative and non-rate-limited (Kugler and Turvey 1987). An adiabatic transformation requires that the rhythmic system's action (energy/frequency) and entropy production remain time-invariant throughout the transformation. The non-conservative adiabatic hypothesis was evaluated through an experiment on human rhythmic hand movements. On each trial, a subject began at a prescribed frequency and then, over a 30 s interval, increased (or decreased) the frequency continuously at will. For each subject, on each increasing and decreasing trial, cycle kinetic energy was a linear function of cycle frequency with a negative energy intercept. By the adiabatic hypothesis, the slope of the function defines the constant action and the intercept defines the constant dissipation — changes in cycle frequency incur no changes in energy dissipated per cycle. Slopes and intercepts were correlated suggesting a common basis for the two constants, and the variety of cycle amplitude-cycle duration relations were in agreement with the nonmonotonic, nonlinear space-time function predicted by the hypothesis. The possibilities of addressing aspects of the data through (a) muscle modeled as a continuum of Kelvin bodies with a continuous relaxation spectrum, and (b) various classes of autonomous differential equations, were discussed. Most importantly, the discussion focused on the puzzling independence of energy cost and speed exhibited by locomoting animals differing in morphology, physiology, size, and taxa. It was suggested that the independence may reflect a very general principle — adiabatic transformability of biological movement systems.     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

Modeling the ecological niche of long-term land use changes: The role of biophysical factors

Land use/land cover changes (LULCCs) represent the result of the complex interaction between biophysical factors and human activity, acting over a wide range of temporal and spatial scales. The aim of this work is to quantify the role of biophysical factors in constraining the trajectories of land abandonment and urbanization in the last 50 years. A habitat suitability model borrowed from animal ecology was used to analyze the ecological niche of the following LULCC trajectories occurred in Emilia-Romagna (northern Italy) during 1954–2008: (i) land abandonment (LA) and (ii) urbanization (URB), both from agricultural areas (URB_agr) and from semi-natural areas (URB_for). Results showed that the different LULCC trajectories were driven by different combinations of biophysical factors, such as climate, topography and soil quality. In particular, slope and elevation resulted as the main driving factors for rural processes, while slope and temperatures resulted as the main constraints underlying urban processes. This approach may represent a conceptual and technical step toward the systematic assessment of LULCC processes, thus providing an effective support tool to inform decision makers about land use transformations, their underlying causes, as well as their possible implications.     

Neurotransmitters regulate rhythmic size changes amongst cells in the fly's optic lobe

Axon calibre in monopolar cells L1 and L2 of the fly's lamina can change dynamically. Swelling by day, L2 exhibits a daily rhythm of changing size apparntly mediated by wide-field LBO5HT and PDH cells. L1/L2 axon profiles were measured planimetrically in the housefly, Musca domestica, from 1 m cross sections. Four hours after injecting 80–100 nl of 1.25 × 10^–4 M 5-HT into the optic lobe, L1's axon swelled but L2's did not, whereas 2.2 × 10^–5 M of PDH enlarged both axons. Similar to 5-HT, 1.63 × 10^–4 M histamine (the photoreceptor transmitter) enlarged L1 but not L2, mimicking light exposure, while 1.7 × 10^–4 M glutamate and 1.94 × 10^–4 M GABA both decreased L1 and L2. 2.5 × 10^–4 M of 5,7-dihydroxytryptamine decreased L2 and, somewhat, L1, an effect attributable to the loss of LBO5HT neurites. Twenty four hours after cutting LBO5HT and PDH commissural pathways, L1 and L2 both shrank. Apparently, L2's size depends on either LBO5HT or sufficient 5-HT, and L1 and L2 have different response ranges to 5-HT. Responses to PDH imply that daytime PDH release drives a circadian rhythm, enlarging L1 and L2.Abbreviations ERG electroretinogram - GABA -aminobutyric acid - PDH pigment-dispersing hormone - PDF pigment-dispersing factor - 5-HT 5-hydroxytryptamine - EM electron microscopy(ic)     

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

Protein structure and dynamics are crucial for protein function. Thus, the study of conformational properties can be very informative for characterizing new proteins and to rationalize how residue substitutions at specific protein sites affect its dynamics, activity and thermal stability. Here, we investigate the structure and dynamics of the recently isolated cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila (SpAAP) by the integration of simulations, circular dichroism, mass spectrometry and other experimental data. Our study notes traits of cold-adaptation, such as lysine-to-arginine substitutions and a lack of disulphide bridges. Cold-adapted enzymes are generally characterized by a higher number of glycine residues with respect to their warm-adapted counterparts. Conversely, the SpAAP glycine content is lower than that in the warm-adapted variants. Nevertheless, glycine residues are strategically located in proximity to the functional sites in SpAAP, such as the active site and the linker between the two domains.. In particular, G457 reduces the steric hindrance around the nucleophile elbow. Our results suggest a local weakening of the intramolecular interactions in the cold-adapted enzyme. This study offers a basis for the experimental mutagenesis of SpAAP and related enzymes. The approaches employed in this study may also provide a more general framework to characterize new protein structures in the absence of X-ray or NMR data.     

TFAR19 gene changes the biophysical properties of murine erythroleukemia cells

TFAR19 is a novel apoptosis-related gene and can accelerate cell apoptosis in the presence of apoptosis inducements. Here, we studied the effects of TFAR19 on some biophysical properties of mouse erythroleukemia (MEL) cells and their molecular and structural basis. After transfected with TFAR19 and apoptosis inducement, MEL revealed a high cell membrane fluidity, a decrease in resynthesis of phospholipids, an increase in the proteins/nucleic acids ratio, a relatively orderly cytoskeleton network, an impaired deformability, a low integrin aM expression, and a decrease in adhesion to endothelial cells. These findings suggest the potential of TFAR19 for antitumor cell migration, and thus for antitumor gene therapy.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.