Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Phospholipid oxidation catalyzed by cytochrome c in liposomes

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.     

Laser light-scattering characterization of mitochondrial complex III-Triton X-100-phospholipid mixed micelles

Bovine-heart mitochondrial complex III was purified in the presence of Triton X-100, and the size and shape of the resulting protein-surfactant-phospholipid mixed micelles were investigated by laser light-scattering. The protein appears to be present in the form of a dimer, irrespective of temperature (between 25 and 40 degrees C) and protein concentration (between 0.5 and 5 mg/ml). The molecular weight of the micelle increases with temperature from 600 000 (25 degrees C) to 692 000 (40 degrees C). The variation of the solvent second virial coefficient in this temperature range suggests that, with increasing temperature, some of the free surfactant molecules become integrated in the mixed micelles. The average quadratic radius of gyration of these is of 42 +/- 5 nm, corresponding in our case to an ellipsoidal shape.     

Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion

Limited digestion of filamentous myosin with chymotrypsin at 0 degrees C in the absence of divalent cations generates two forms of subfragment 1 (S1), with heavy chains of 95 kDa and 98 kDa. The difference is at the C-terminal end of the chain. The 98 kDa form prevails, in contrast to the preparations obtained by digestion at room temperature which consist of the shorter species and only traces of the longer one. The results support the idea of a temperature-dependent conformational transition at the head-rod junctional region of the myosin heavy chain.     

Application of fuzzy multiobjective optimization for self-sucking impellers in a bioreactor

For three types of self-sucking impellers (fourand six-pipe and disk impellers) mixing power, initial point, amount of gas leaving the impeller and mass transfer coefficient were determined experimentally. Investigations were performed for two systems: water and biomass solution.From the point of view of a minimum mixing power and maximum mass transfer coefficient the best impeller has been chosen. Fuzzy multiobjective optimization for determination of optimum operating conditions is proposed.List of Symbols c concentration of oxygen - D tank diameter - d impeller diameter - g acceleration of gravity - H height of liquid in the tank - H height of liquid above impeller, H=H-y - k consistency coefficient - k _L a volumetric mass transfer coefficient - N rotational speed of impeller - n flow behaviour index - P mixing power for pure liquid - P _G mixing power for aerated liquid - V _G volumetric air flow rate - y distance of impeller from the tank bottom - v _a apparent kinematic viscosity of liquid - density of liquid - time - gas hold-up - Eu=P/N ³ d ⁵ or Eu_G=P _G /N ³ d ⁵ Euler Number for non-gassed or aerated liquid - Fr=N ² d/g Froude Number - Fr^*=N ² d ² /g(H -y) modified Froude Number - K_G=V _G /N d ³ gas flow number - Re=N d ² /v _a Reynolds Number - Sh=k _K a/(g ² /v _a )^1/3 Sherwood Number     

An electrogenetic toggle switch model

Synthetic biology uses molecular biology to implement genetic circuits that perform computations. These circuits can process inputs and deliver outputs according to predefined rules that are encoded, often entirely, into genetic parts. However, the field has recently begun to focus on using mechanisms beyond the realm of genetic parts for engineering biological circuits. We analyse the use of electrogenic processes for circuit design and present a model for a merged genetic and electrogenetic toggle switch operating in a biofilm attached to an electrode. Computational simulations explore conditions under which bistability emerges in order to identify the circuit design principles for best switch performance. The results provide a basis for the rational design and implementation of hybrid devices that can be measured and controlled both genetically and electronically.     

MHC zygosity in the frog,Rana temporaria

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.     

Helix-forming tendencies of amino acids depend on the restrictions of side-chain rotamer conformations: crystal structure of the tripeptide GAI in two crystalline forms.

In our attempts to design crystalline alpha-helical peptides, we synthesized and crystallized GAI (C11H21N3O4) in two crystal forms, GAI1 and GAI2. Form 1 (GAI1) Gly-L-Ala-L-Ile (C11H21N3O4.3H2O) crystals are monoclinic, space group P2(1) with a = 8.171(2), b = 6.072(4), c = 16.443(4) A, beta = 101.24(2) degrees, V = 800 A3, Dc = 1.300 g cm-3 and Z = 2, R = 0.081 for 482 reflections. Form 2 (GAI2) Gly-L-Ala-L-Ile (C11H21N3O4.1/2H2O) is triclinic, space group P1 with a = 5.830(1), b = 8.832(2), c = 15.008(2) A, alpha = 102.88(1), beta = 101.16(2), gamma = 70.72(2) degrees, V = 705 A3, Z = 2, Dc = 1.264 g cm-3, R = 0.04 for 2582 reflections. GAI1 is isomorphous with GAV and forms a helix, whereas GAI2 does not. In GAI1, the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an incipient alpha-helix. GAI2 imitates a cyclic peptide and traps a water molecule. The conformation angles chi 11 and chi 12 for the side chain are (-63.7 degrees, 171.1 degrees) for the helical GAI1, and (-65.1 degrees, 58.6 degrees) and (-65.0 degrees, 58.9 degrees) for the two independent nonhelical molecules in GAI2; in GAI1, both the C gamma atoms point away from the helix, whereas in GAI2 the C gamma atom with the g+ conformation points inward to the helix and causes sterical interaction with atoms in the adjacent peptide plane. From these results, it is clear that the helix-forming tendencies of amino acids correlate with the restrictions of side-chain rotamer conformations. Both the peptide units in GAI1 are trans and show significant deviation from planarity [omega 1 = -168(1) degrees; omega 2 = -171(1) degrees] whereas both the peptide units in both the molecules A and B in GAI2 do not show significant deviation from planarity [omega 1 = 179.3(3) degrees; omega 2 = -179.3(3) degrees for molecule A and omega 1 = 179.5(3) degrees; omega 2 = -179.4(3) degrees for molecule B], indicating that the peptide planes in these incipient alpha-helical peptides are considerably bent.