Thermodynamics of neutral protein evolution

Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (delta deltaG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the single-mutation delta deltaG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.     

EDTA-induced Membrane Fluidization and Destabilization： Biophysical Studies on Artificial Lipid Membranes

The molecular mechanism of ethylenediaminetetraacetic acid （EDTA）-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine （DPPC） or mixtures of DPPC and metal-chelating lipids, such as N^a,N^a-Bis[carboxymethyl]-N^ε [（dioctadecylamino）succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-（5-amino- 1 -carboxypentyl iminodiacetic acid） succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.     

Constitutive secretion of β-trace protein by cultivated porcine choroid plexus epithelial cells: Elucidation of its complete amino acid and cDNA sequences

Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of β-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. β-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human β-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian β-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of β-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid. © 1996 Wiley-Liss, Inc.     

Fruktifikationen der Gattung Hemitrapa Miki (Trapellaceae) in den Ablagerungen der Oberen Süßwasser-Molasse Bayerns (mit Bemerkungen zu den fossilen Vorkommen Eurasiens)

In contrast to the opinion of Miki (1952 b, p. 349) the genus Hemitrapa Miki (Trapellaceae) is not only found in Asia and America but also in Europe. Wrongly determined fossils of the type „Trapa silesiaca Goeppert”︁ (the original species from Schoßnitz in Poland is under research by M. Lancucka-Srodoniowa, Krakow) belong to the genus Hemitrapa Miki. Some other Bavarian fossils are newly decribed here as Hemitrapa heissigii sp. nov. Hemitrapa fossils grew not only in the Senftenberg area (Menzel 1906), in Silesia (Kräusel 1920) or the Niederlausitz area (Menzel in Gothan & Sapper 1933), at Konin (Raniecka-Bobrowska 1954), but possibly also near Cologne (Kilpper 1969 and Kramer 1974) and at Ponholz (Gregor 1980). Especially in the Middle Miocene Upper Freshwater Molasse of Bavaria the fossil species Hemitrapa heissigii is found in numerous specimens near Eberstetten, Haag a. d. Amper and Rauscheröd (all in southern Bavaria). Hemitrapa heissigii can be used as an index-fossil and signs Uppermost Miocene sediments (Badenian, Samartian). The occurence in Pliocene localities is to be prooved. The whole group around Hemitrapa is considered to be a “late element” (Upper Miocene) in Europe.     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

A Common Structural Motif in Elongation Factor Ts and Ribosomal Protein L7/12 May Be Involved in the Interaction with Elongation Factor Tu

Elongation factor (EF) Tu alternates between two interaction partners, EF-Ts and the ribosome, during its functional cycle. On the ribosome, the interaction involves, among others, ribosomal protein L7/12. Here we compare EF-Ts and L7/12 with respect to the conservation of sequence and structure. There is significant conservation of functionally important residues in the N-terminal domain of EF-Ts and in the C-terminal domain of L7/12. The structure alignment based on the crystal structures of the two domains suggests a high degree of similarity between the αA–βD–αB motif in L7/12 and the h1–turn–h2 motif in EF-Ts which defines a common structural motif. The motif is remarkably similar with respect to fold, bulkiness, and charge distribution of the solution surface, suggesting that it has a common function in binding EF-Tu. Received: 12 June 2000 / Accepted: 10 October 2000     

Automated multi-well device to measure transepithelial electrical resistances under physiological conditions

Measurement of transendothelial or transepithelial electrical resistances (TERs) is a straightforward in situ experimental approach to monitor the expression or modulation of barrier-forming cell-to-cell contacts (tight junctions) in cultured cells grown on porous filters. Although widely accepted, there is currently no device available to automatically measure the time course of TERs under ordinary cell culture conditions (37 degrees C, 5% or 10% CO2). This paper describes a development from our laboratory that is capable of following in parallel the TERs of several filter-grown cell layers with time and in an entirely computer-controlled fashion. The cell cultures can be followed even in long-term experiments without any manual assistance or opening of the incubator Besides reading TER values, this approach also returns the electrical capacitance of the cell layers, which is indicative of the expression of microvilli and other membrane extrusions. The device is based on reading the frequencydependent impedance of the cell layer, followed by equivalent circuit modeling to extract the cell-related parameters. It is compatible with several multi-well formats (up to 96 wells) and controlled by custom-designed software that reads, analyzes, and presents the data.     

Anti-angiogenic effects of thalidomide: expression of apoptosis-inducible active-caspase-3 in a three-dimensional collagen gel culture of aorta

The anti-angiogenic properties of thalidomide have led to the use of the agent as a remedy for multiple myeloma. Nevertheless, the anti-angiogenic moiety of thalidomide remains unidentified. In this study we examined the anti-angiogenic effects of thalidomide in an in vitro model using a three-dimensional collagen gel culture. Angiogenesis was significantly inhibited when the culture was treated with thalidomide plus cytochrome P-450 (CYP2B4), and the migrating cells and tubules were positive for active-caspase-3 in an accompanying immunohistochemical investigation. Transmission electron microscopic observation also confirmed that active-caspase-3-positive cells demonstrated apoptotic characteristics. This study is the first to morphologically demonstrate the effect of thalidomide in directly inducing the apoptosis of new tubules and migrating cells on a three-dimensional collagen gel culture of aorta. Taken together with earlier findings, our new results indicate that the thalidomide-induced inhibition of angiogenesis involves apoptosis in addition to the suppression of TNF- and inhibition of cell migration from aorta explants, i.e., the factors important for capillarogenesis.     

Absence of intrinsic electric conductivity in single dsDNA molecules

The intrinsic dc conductivity of long, individual lambda phage dsDNA molecules has been investigated by ultrasensitive low current-voltage-spectroscopy (IV) under ambient conditions and controlled low humidity inert gas atmosphere on microfabricated metal-insulator-metal gap structures. We found a strong dependence of the measured conductivity on the apparent humidity, which we attribute to capillary condensation of water to the immobilized DNA molecules, giving rise to additional ionic currents. Additional IV-spectroscopy experiments under controlled argon atmosphere always revealed a significant drop in electrical conductivity to 4 x 10(-15)AV(-1)microm(-1), indicating almost no considerable contribution of electrical long range charge transport.