Nonnative protein polymers: structure, morphology, and relation to nucleation and growth

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10³-10⁵ kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (M_w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M_w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (τ_n) and growth (τ_g), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ_n, τ_g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ_n/τ_g and aggregate M_w.     

Surface colony growth in a controlled nutrient environment. 3. Dependence of growth constant on nutrient concentration.

The growth rates of yeast surface colonies under constant nutrient conditions were studied as a function of nutrient concentration. This was complementary to the initial experiment to measure the rates under such conditions, and to the investigation of the effects of alcohol. The possibility of fitting an equation of the Michaelis-Menten type to the variation of growth rate with carbon source concentration is considered.     

Microtubule oscillations. Role of nucleation and microtubule number concentration

Microtubules are capable of performing synchronized oscillations of assembly and disassembly which has been explained by reaction mechanisms involving tubulin subunits, oligomers, microtubules, and GTP. Here we address the question of how microtubule nucleation or their number concentration affects the oscillations. Assembly itself requires a critical protein concentration (Cc), but oscillations require in addition a critical microtubule number concentration (CMT). In spontaneous assembly this can be achieved with protein concentrations Cos well above the critical concentration Cc because this enhances the efficiency of nucleation. Seeding with microtubules can either generate oscillations or suppress them, depending on how the seeds alter the effective microtubule number concentration. The relative influence of microtubule number and total protein concentrations can be varied by the rate at which assembly conditions are induced (e.g. by a temperature rise): Fast T-jumps induce oscillations because of efficient nucleation, slow ones do not. Oscillations become damped for several reasons. One is the consumption of GTP, the second is a decrease in microtubule number, and the third is that the ratio of microtubules in the two phases (growth-competent and shrinkage-competent) approach a steady state value. This ratio can be perturbed, and the oscillations restarted, by a cold shock, addition of seeds, addition of GTP, or fragmentation. Each of these is equivalent to a change in the effective microtubule number concentration.     

Structures and magnetic properties of one-dimensional polymers constructed with copper(II) salts and pyridinecarboxamide-type ligands

Three complexes of the composition {[Cu(μ_1,5-dca)₂(mppca)₂] · H₂O}_n (1), [Cu(μ_1,5-dca)₂(nppca)₂]_n (2) and [Cu(μ-Cl)₂(mppca)₂]_n (3) (dca = dicyanamide, ; mppca = N-(4′-methylphenyl)-4-pyridinecarboxamide; nppca = N-(4′-nitrophenyl)-4-pyridinecarboxamide) have been synthesized and characterized by single crystal X-ray crystallography and magnetic susceptibility studies. Different supramolecular structures of the complexes have been constructed by different non-covalent motifs in the crystalline solids. In complex 1, adjacent copper(II) atoms are connected by double μ_1,5-dca(end-to-end) bridges to form a chain-like structure. The chains are linked by π-π interactions and hydrogen bonds between the ligands and water molecules to form a 3D network. In complex 2, copper(II) atom has a coordination environment similar to 1, but water molecules have not been found. Weak C-H?N hydrogen bonding and π-π interaction yield a 3D supramolecular network which is different from that of complex 1. Complex 3 is a 1D polymeric chain in which Cu(II) ions are bridged by Cl⁻, and only CH/π interactions had been found. Magnetic measurements revealed antiferromagnetic properties of 1, 3 and ferromagnetic behavior of 2.     

Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport.

Assembly and motor activity of myosin II affect shape, contractility, and locomotion of nonmuscle cells. We used fluorescent analogues and imaging techniques to elucidate the state of assembly and three-dimensional distribution of myosin II in living Swiss 3T3 fibroblasts. An analogue of myosin II that was covalently cross-linked in the 10S conformation and unable to assemble served as an indicator of the cytoplasmic volume accessible to 10S myosin II. Ratio-imaging of an analogue that can undergo 10S-->6S conversion versus the volume indicator revealed localized concentration of assembly-competent myosin II. In stationary serum-deprived cells and in cells locomoting at the edge of a wound, it was most concentrated in the peripheral cytoplasm, where fibers containing myosin II assemble, and least concentrated in the perinuclear cytoplasm, where they disassemble. Furthermore, fluorescence photobleaching recovery showed myosin II to be less mobile in the periphery than in perinuclear cytoplasm. These results indicate a gradient in the assembly of myosin II. Three-dimensional microscopy of living cells revealed that fibers containing myosin II were localized in the cortical cytoplasm, whereas myosin II was diffusely distributed in the deeper cytoplasm, suggesting that myosin II is assembled preferentially near the cell surface. Localized protein phosphorylation may play a role, because a kinase inhibitor, staurosporine, abolished the gradient of myosin II assembly.     

Vesicular cholesterol in bile. Relationship to protein concentration and nucleation time

A study was done to determine whether the nucleation time was related to the amount of cholesterol carried in vesicles. Bile was obtained from cholesterol gallstone patients and controls. Gel-exclusion chromatography was used to separate vesicles and micelles in the native bile using an eluting buffer containing 10 mM sodium cholate. The percent of total cholesterol carried in vesicles in gallbladder bile of stone patients was significantly greater than that in control patients. Total cholesterol concentration in gallbladder bile of stone patients was significantly greater than in controls. This difference was due to the fact that vesicular cholesterol concentration was significantly greater in the gallbladder bile of stone patients compared to controls. Micellar cholesterol concentrations were similar in the two groups. Nucleation time was related significantly to vesicular cholesterol concentration in correlation analysis and, as previously shown, so was total protein concentration. This study supports the importance of vesicular cholesterol in solid crystal formation and demonstrates for the first time that the rate of cholesterol monohydrate crystal formation is directly related to the amount of cholesterol transported in vesicles.     

Growth of Qinghai Tibetans living at three different high altitudes

This study compares the stature, weight, skinfolds, upper arm muscle area, and chest dimensions of Tibetan children, adolescents, and young adults who were born and raised, or who had lived from infancy, at 3,200 m, 3,800 m, and 4,300 m in Qinghai Province, People's Republic of China. While the individuals measured in Qinghai are among the tallest and heaviest Tibetans reported in the literature, they are nevertheless smaller and lighter than well-off children living at low altitude. The pattern of size variation among Tibetan males and females measured at the three high altitudes, along with evidence of a secular trend at 4,300 m, suggests that nutrition may significantly effect growth at high altitude. Only minor differences in thorax dimensions exist between Tibetan males and females measured at 3,200 m and 3,800 m. However, Tibetan males at 4,300 m possess slightly narrower and deeper chests (during and after adolescence) than males at 3,200 m and 3,800 m. Since individuals from 3,800 m and 4,300 m belong to the same local populations, this characteristic is unlikely to be genetically determined. However, it may be related to differences in the degree of hypoxia or to the influences of other environmental conditions.     

Simulations of nucleation and early growth stages of protein crystals.

Analysis of known protein crystal structures reveals that interaction energies between monomer pairs alone are not sufficient to overcome entropy loss related to fixing monomers in the crystal lattice. Interactions with several neighbors in the crystal are required for stabilization of monomers in the lattice. A microscopic model of nucleation and early growth stages of protein crystals, based on the above observations, is presented. Anisotropy of protein molecules is taken into account by assigning free energies of association (proportional to the buried surface area) to individual monomer-monomer contacts in the lattice. Lattice simulations of the tetragonal lysozyme crystal based on the model correctly reproduce structural features of the movement of dislocation on the (110) crystal face. The dislocation shifts with the speed equal to the one determined experimentally if the geometric probability of correct orientation is set to 10(-5), in agreement with previously published estimates. At this value of orientational probability, the first nuclei, the critical size of which for lysozyme is four monomers, appear in 1 ml of supersaturated solution on a time scale of microseconds. Formation of the ordered phase proceeds through the growth of nuclei (rather then their association) and requires nucleations on the surface at certain stages.     

On Darwin's 'metaphysical notebooks'. II: "Metaphysics" and final cause.

The first part of this paper was published in Rivista di Biologia/Biology Forum 94 (2001). In the second part below an examination is made of the meaning of the term Metaphysics in some passages of the Darwinian Notebooks for the years 1836-1844. Metaphysics no longer defines a field of philosophical enquiries mainly concerning the being and the essence after the manner of Aristotle; it now refers to a kind of philosophy of mind after the manner of J. Locke's criticism of the Hypokeimenon. However Aristotle's Metaphysics also encompasses a treatment of the idea of causes, and of final cause particularly, in the explanation of events, and in the explanation of natural phenomena especially. The criticism of the idea of final cause in the interpretation of the world of life is one of Darwin's foundational acts in his early years. When conceiving his Système du monde, in the last years of the XVIII Century, Laplace could think that God is a hypothesis not really needed by science, as we are told. For the knowledge of organic nature to attain the status of science, it remained to be shown that since--certain of the exemplariness of Newton's Principles as much as cautious before the mystery of life--did not need the hypothesis of final ends in order to understand and explain the productions of the living nature: not only in the form of that final cause (the First Cause, the Vera Causa) in which Natural Theology still rested, but also in the form of nature's inner finality which still moulded Whewell's Kantian philosophy. Such demonstration is a very important subject in Darwin's early enquiries, where he criticises finalism as a projection of self-conceiving Man, likely inherited from a knowing of causality in nuce to be found also in animals.     

The modelled growth of mycorrhizal and non-mycorrhizal plants under constant versus variable soil nutrient concentration

We studied the response of mycorrhizal and non-mycorrhizal plants to variation in soil nutrient concentration. A model for the relative growth rate (RGR) of plant biomass was constructed with soil nutrients as an explanatory variable. A literature survey was carried out to find the relative magnitudes of parameter values for mycorrhizal and non-mycorrhizal plants. Mycorrhizal plants had higher RGR at low nutrient concentrations and non-mycorrhizal plants at high nutrient concentrations. The RGR of mycorrhizal and non-mycorrhizal plants at constant versus log-normally distributed soil nutrient concentration were compared to see the effect of mycorrhizal status on responses to variation. Variation in nutrient concentration generally reduced RGR, especially in mycorrhizal plants. The RGR of a non-mycorrhizal plant may increase with variation where a growth function threshold exists, i.e. a soil nutrient concentration that must be exceeded to allow growth. Mycorrhizal plants appeared more sensitive to variation in nutrient concentration than non-mycorrhizal plants due to the higher affinity of mycorrhizal roots at low nutrient levels. However, this prediction may be reversed if mycorrhizal symbiosis considerably stabilises flow of nutrients to plant physiological processes, such that mycorrhizal plants experience less variation in soil nutrient concentration than non-mycorrhizal plants. Our results also attain broader significance by suggesting a general trade-off between competitive ability in a constant versus variable resource availability.     

Two cobalt(II) one-dimensional ribbon of rings polymers: Syntheses, structures and non-linear optical properties

Two cobalt(II) coordination polymers {[Co(SCN)₂(3-bpit)₂] · 2CH₃OH}_n (1) (3-bpit = N,N′-bis(3-pyridylformyl)imidazolidine-2-thione) and {[Co(SCN)₂(4-bpit)₂] · CH₃OH · CH₃CN}_n (2) (4-bpit = N,N′-bis(4-pyridylformyl)imidazolidine-2-thione) have been synthesized for study of the third-order non-linear properties. X-ray crystal structural analyses reveal that the two polymers show the same topological motif: a ribbon of rings. Their third-order non-linear optical (NLO) properties in DMF solution have been studied by Z-scan techniques with a linearly polarized laser light (λ = 532 nm; pulse widths = 7 ns). Both of them exhibit strong self-focusing effect. The third-order NLO susceptibility χ⁽³⁾ values are calculated to be 2.61 × 10⁻¹¹ esu (1) and 2.76 × 10⁻¹¹ esu (2), respectively. The values are comparable to those of the reported good NLO materials.