An innovative approach for testing bioinformatics programs using metamorphic testing

Background  

Recent advances in experimental and computational technologies have fueled the development of many sophisticated bioinformatics programs. The correctness of such programs is crucial as incorrectly computed results may lead to wrong biological conclusion or misguide downstream experimentation. Common software testing procedures involve executing the target program with a set of test inputs and then verifying the correctness of the test outputs. However, due to the complexity of many bioinformatics programs, it is often difficult to verify the correctness of the test outputs. Therefore our ability to perform systematic software testing is greatly hindered.     

Dielectrophoresis and electrorotation of neurospora slime and murine myeloma cells.

Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes.     

Electroporation of cell membranes.

Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given.     

Reversible mechanosensitive ion pumping as a part of mechanoelectrical transduction.

To explain the ability of some mechanosensitive cells to reverse the process of mechanotransduction and to generate mechanical oscillations and emit sound, a piezo-conformational coupling model (PCC model) is proposed. The model includes a transport protein which changes either its volume (PV-coupling) or its area in the membrane (gamma A-coupling) when undergoing conformational transitions. Such a protein can interact with an oscillating pressure to pump ions and create a transmembrane gradient if the affinities of the protein for ions are different at the two sides of membrane. The frequency and concentration windows for mechanical energy transduction were determined. Under optimal conditions, the efficiency of energy transduction can approach the theoretical maximum of 100%. If the concentration gradient exceeds the static head value (quasi-equilibrium which can be built up and maintained by this transport system), the energy transduction reverses and the transporter becomes a generator of mechanical oscillations at the expense of a concentration gradient. Estimation of thermodynamic parameters of the pump shows that the PV-coupling model would require large pressure oscillations to work while the gamma A-coupling model could work in physiological conditions. The gamma A-coupling mechanism may be used by cells for two purposes. In the reverse mode, it can be a force generator for various applications. In the direct mode, it may serve bioenergetic purposes by harvesting the energy of mechanical oscillations and storing it in the form of a concentration gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

A study on the stability of an estrogen-protein conjugate in vivo and under in vitro conditions

[³ H] Estradiol-17β-succinyl bovine serum albumin conjugate ([³H]-E₂-BSA) was synthesized with a specific activity of 1.92 × 10⁷ cts/min/mg. The conjugate was administered iv to ovariectomized rats and the quantity of free [³H] steroid in the uterus was determined. Radioactive material was detected in all of the subcellular fractions of the uterus and identified as estradlol-17β. Similar subcellular distribution of the radioactivity was observed when [³H]E₂-BSA was added $\text{in vitro}$ to uterine homogenate. Free estradlo1-17β was released when the conjugate was Incubated with rat uterine homogenate or with serum. The results of the present study suggest that E₂-BSA is hydrolyzed $\text{in vivo}$ and under $\text{in vitro}$ conditions. It is recommended that the stability of a hormone-protein conjugate be established before use.     

Local hydrophobicity stabilizes secondary structures in proteins

The probability of occurrence of helix and β-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor β-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor β-sheet formation with hydrophilic neighbors, but most of them strongly favor β-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and β-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for β-sheets. Therefore, β-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein–protein and protein–lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.     

Comparative study of an adenosine triphosphatase trigger-fused lipid vesicle and other vesicle forms of dimyristoylphosphatidylcholine

Several known forms of bilayer vesicles of dimyristoylphosphatidylcholine exhibit the gel to liquid-crystalline phase transition in the temperature range convenient for membrane enzyme reconstitution studies. This warrants a systematic investigation of their physical characteristics and their phase transition behaviors. We have employed electron microscopy, gel chromatography, 31P nuclear magnetic resonance, differential scanning microcalorimetry, and fluorescence spectroscopy to determine several physical parameters of the limiting size microvesicle (260 +/- 40 A), the larger vesicle form (900 +/- 100A) of Enoch and Strittmatter [Enoch, H. G., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145], the multilamellar vesicle, and, in particular, an ATPase-trigger-fused macrovesicle (950 +/- 200 A). This latter vesicle form was produced by a spontaneous fusion of the complex of the plasma membrane ATPase of Schizosaccharomyces pombe and the lipid microvesicles at a low ratio of enzyme to vesicle concentrations, and at a low temperature (around 10 degrees C). The ATPase-trigger-fused vesicles are unilamellar and have an intact ionic permeation barrier at 30 degrees C and a gel to liquid-crystalline transition temperature at 24.4 degrees C with a transition heat of 5.64 kcal/mol. Thus, this vesicle form should be a valuable tool for studying possible proton-pumping activity of this ATPase. In contrast to data found in the literature, which show lack of the pretransition for unilamellar microvesicles, we have observed the pretransition around 15 degrees C for all the vesicle forms examined. Moreover, the transition widths of unilamellar vesicles are much broader than those of the multilamellar vesicles, suggesting that in the latter system interlayer interactions may contribute to the cooperativity of the transition.     

High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures

High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60 degrees C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the delta Hcal (calorimetric enthalpy) of denaturation (1715 +/- 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, within experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 +/- 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observed functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60 degrees C, with an delta Hcal of 1058 +/- 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 +/- 0.1 degrees C with an delta Hcal of 255 +/- 12 kcal/mol. Light chains melted at 51.0 +/- 0.2 degrees C with an delta Hcal of 85 +/- 15 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)