Metabolic network alignment in large scale by network compression

Metabolic network alignment is a system scale comparative analysis that discovers important similarities and differences across different metabolisms and organisms. Although the problem of aligning metabolic networks has been considered in the past, the computational complexity of the existing solutions has so far limited their use to moderately sized networks. In this paper, we address the problem of aligning two metabolic networks, particularly when both of them are too large to be dealt with using existing methods. We develop a generic framework that can significantly improve the scale of the networks that can be aligned in practical time. Our framework has three major phases, namely the compression phase, the alignment phase and the refinement phase. For the first phase, we develop an algorithm which transforms the given networks to a compressed domain where they are summarized using fewer nodes, termed supernodes, and interactions. In the second phase, we carry out the alignment in the compressed domain using an existing network alignment method as our base algorithm. This alignment results in supernode mappings in the compressed domain, each of which are smaller instances of network alignment problem. In the third phase, we solve each of the instances using the base alignment algorithm to refine the alignment results. We provide a user defined parameter to control the number of compression levels which generally determines the tradeoff between the quality of the alignment versus how fast the algorithm runs. Our experiments on the networks from KEGG pathway database demonstrate that the compression method we propose reduces the sizes of metabolic networks by almost half at each compression level which provides an expected speedup of more than an order of magnitude. We also observe that the alignments obtained by only one level of compression capture the original alignment results with high accuracy. Together, these suggest that our framework results in alignments that are comparable to existing algorithms and can do this with practical resource utilization for large scale networks that existing algorithms could not handle. As an example of our method's performance in practice, the alignment of organism-wide metabolic networks of human (1615 reactions) and mouse (1600 reactions) was performed under three minutes by only using a single level of compression.     

The phase behavior of lipid monolayers containing pulmonary surfactant protein C studied by fluorescence light microscopy

Three compounds of the pulmonary surfactant – dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant associated protein C (SP-C) – were spread at the air-water interface of a Langmuir trough as a model system to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity, the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant to stability in lungs during expiration and inhalation. Received: 13 February 1997 / Accepted: 22 May 1997     

Functional molecular size of trypsin inhibitors as determined by radiation inactivation analysis

The molecular sizes of trypsin inhibitors obtained by radiation inactivation were investigated in relation to the functional domain size. The molecular weights obtained were 10,200 +/- 700 for ovomucoid, 17,800 +/- 400 for ovoinhibitor and 16,400 +/- 500 for soybean trypsin inhibitor. These values mostly agreed with the size of domain which has trypsin inhibitory activity, suggesting that the radiation inactivation analysis indicates the minimum functional domain size.     

Compression of fractionated sun-cured and dehydrated alfalfa chops into cubes--specific energy models

The objective of this study was to determine the specific energy requirements for the compression of fractionated sun-cured and dehydrated alfalfa chops, when subjected to different pressures and holding times. The compression behavior of fractionated sun-cured and dehydrated alfalfa chops was studied using a single cubing unit capable of making one cube in a single stroke of the plunger. The cube die dimensions were 30 mm x 30 mm in cross-section and an effective depth of compression of 0.38 m. The initial moisture content of dehydrated and sun-cured chops were 6% and 7% (wb), respectively. A stack of two sieves (instead of five) was used along with a pan to achieve leaf and stem separation. The nominal opening sizes of two sieves with square holes were 3.96 and 1.17 mm, respectively. Leaf and stem fractions were combined later to obtain five different samples each for sun-cured and dehydrated alfalfa with leaf content ranging from 0% to 100% by mass in increments of 25%. The chop moisture content and preheat temperature before compaction was 10% (wb) and 75 degrees C, respectively. The cube die temperature was maintained at 90+/-5 degrees C. The mass of chops used for making each cube was 23+/-02 g. A hydraulic press was used to apply 9.0, 12.0 and 14.0 MPa of pressures through a plunger. After compression, the plunger was held in place for 10 and 30s, before the compacted forage was extracted. Empirical equations were fitted to the data relating specific energy for cube making to pressure, residence time, and leaf content.     

Investigating the Effect of Particle Size on Pulmonary Surfactant Phase Behavior

We study the impact of the addition of particles of a range of sizes on the phase transition behavior of lung surfactant under compression. Charged particles ranging from micro- to nanoscale are deposited on lung surfactant films in a Langmuir trough. Surface area versus surface pressure isotherms and fluorescent microscope observations are utilized to determine changes in the phase transition behavior. We find that the deposition of particles close to 20 nm in diameter significantly impacts the coexistence of the liquid-condensed phase and liquid-expanded phase. This includes morphological changes of the liquid-condensed domains and the elimination of the squeeze-out phase in isotherms. Finally, a drastic increase of the domain fraction of the liquid-condensed phase can be observed for the deposition of 20-nm particles. As the particle size is increased, we observe a return to normal phase behavior. The net result is the observation of a critical particle size that may impact the functionality of the lung surfactant during respiration.     

Effect of line tension on the lateral organization of lipid membranes

The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes.     

The 3-D model: Comparison of parameters obtained from and by simulating different tableting machines

The aim of this study is to apply 3-D modeling to data obtained from different tableting machines and for different compression wheels on a linear rotary tableting machine replicator. A new analysis technique to interpret these data by 3-D parameter plots is presented. Tablets were produced on an instrumented eccentric tableting machine and on a linear rotary tableting machine replicator. The materials used were dicalcium phosphate dihydrate (DCPD), spray-dried lactose, microcrystalline cellulose (MCC), hydroxypropyl methylcellulose (HPMC), and theophylline monohydrate. Tableting was performed to different maximum relative densities (ρ _{rel, max}). Force, time and displacement were recorded during compaction. The 3-D data plots were prepared using pressure, normalized time, and porosity according to Heckel. A twisted plane was fitted to these data according to the 3-D modeling technique. The resulting parameters were analyzed in a 3-D parameter plot. The results show that the 3-D modeling technique can be applied to compaction cycles from different tableting machines as different as eccentric and rotary tableting machines (simulated). The relation of the data to each other is the same even when the absolute values are different. This is also true for different compression wheels used on the linear rotary tableting machine replicator. By using compression wheels of different sizes on this simulator, mainly time plasticity changes. By using bigger compression wheels for simulation, the materials deform slower at lower densification and they deform faster at higher densification. For brittle materials, the stages of higher densification are influenced; for plastically deforming materials, the stages of lower and higher densification can be influenced.     

Line tension and stability of domains in cell-adhesion zones mediated by long and short receptor-ligand complexes

Submicron scale domains of membrane-anchored receptors play an important role in cell signaling. Central questions concern the stability of these microdomains, and the mechanisms leading to the domain formation. In immune-cell adhesion zones, microdomains of short receptor-ligand complexes form next to domains of significantly longer receptor-ligand complexes. The length mismatch between the receptor-ligand complexes leads to membrane deformations and has been suggested as a possible cause of the domain formation. The domain formation is a nucleation and growth process that depends on the line tension and free energy of the domains. Using a combination of analytical calculations and Monte Carlo simulations, we derive here general expressions for the line tension between domains of long and short receptor-ligand complexes and for the adhesion free energy of the domains. We argue that the length mismatch of receptor-ligand complexes alone is sufficient to drive the domain formation, and obtain submicron-scale minimum sizes for stable domains that are consistent with the domain sizes observed during immune-cell adhesion.     

Investigating the Effect of Particle Size on Pulmonary Surfactant Phase Behavior

We study the impact of the addition of particles of a range of sizes on the phase transition behavior of lung surfactant under compression. Charged particles ranging from micro- to nanoscale are deposited on lung surfactant films in a Langmuir trough. Surface area versus surface pressure isotherms and fluorescent microscope observations are utilized to determine changes in the phase transition behavior. We find that the deposition of particles close to 20 nm in diameter significantly impacts the coexistence of the liquid-condensed phase and liquid-expanded phase. This includes morphological changes of the liquid-condensed domains and the elimination of the squeeze-out phase in isotherms. Finally, a drastic increase of the domain fraction of the liquid-condensed phase can be observed for the deposition of 20-nm particles. As the particle size is increased, we observe a return to normal phase behavior. The net result is the observation of a critical particle size that may impact the functionality of the lung surfactant during respiration.     

Cryptic differentiation and geographic variation in genetic diversity of Hall's Babbler Pomatostomus halli

Sequence variation was examined in domain I of the mitochondrial control region in three Queensland populations of Hall's Babbler Pomatostomus halli , a geographically restricted, monotypic songbird in eastern Australia. Surprisingly, we found that domain I sequences were strongly differentiated into two major clades differing by 3.29%. These two clades exhibited nearly complete geographic concordance with northern and southern populations, except for two haplotypes which were sampled in the north of the range but were phylogenetically allied to the southern clade. We also found a seven-fold higher level of genetic diversity in the northern than in the southern populations. Neutrality and molecular clock tests suggested that selection or differences in substitution rates were not responsible for this difference in diversity. However, a maximum likelihood analysis of gene flow between the north and south suggested that the difference in diversity could be due to both greater population size in the north and asymmetric gene flow dominated by south to north dispersal events. A likelihood ratio test rejected a model in which population sizes were equal and rates of gene flow symmetric, and came close to rejecting a model in which only population sizes were constrained to be equal. These results suggest that different population sizes and asymmetric gene flow could be a major source of differences in genetic variation between populations of Hall's Babbler, although ecological and biogeographic causes for these differences are obscure.     

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1–189 and 190–243) and mouse apoA-I (residues 1–186 and 187–240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.     

Different degrees of lever arm rotation control myosin step size

Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated approximately 90 degrees rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.     

A new epidermal growth factor-like domain in the human core protein for the large cartilage-specific proteoglycan. Evidence for alternative splicing of the domain

A series of cDNA clones for the human core protein of the large cartilage-specific proteoglycan was isolated. Nucleotide sequencing of the clones provided over 2 kilobases of new coding sequences for the human protein. Comparison with published data for cDNA clones covering the same region in rat and chick indicated that domain 8, the lectin-like domain, is highly conserved among species. In contrast, domain 7 is poorly conserved among species. Some of the cDNA clones also contained an additional structural domain between domains 7 and 8 which was not described in the rat or chick sequences. The additional domain of 38 amino acids was highly homologous to epidermal growth factor (EGF)-like sequences seen in other proteins. Because some cDNA clones contained codons for the EGF-like domain and some did not, the results suggested that the EGF-like domain underwent alternative RNA splicing. To confirm alternative splicing of the EGF-like domain, RNA from cartilage cells was used as a template for the polymerase chain reaction. Products of two sizes were obtained. One had the size predicted for mRNA containing the domain and the other had the size predicted for mRNA not containing the domain. Alternative splicing of an EGF-like domain may provide a mechanism of feedback regulation for both the biosynthetic activity and the proliferation of cartilage cells.     

Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers-phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ(av) were quantified for a range of quencher concentrations. Applying the Stern-Volmer formalism, apparent quenching rate constants k(q) were determined from the dependencies on quencher concentration of the ratio τ(0)(av)/τ(av), where τ(0)(av) is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k(q)'/k(q), i.e., the apparent quenching rate constants determined in aggregates (or membranes), k(q)', and in detergent-solubilised LHCII trimers, k(q), respectively. In LHCII macroaggregates, the resulting values for the domain size were 15-30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12-24 LHCII trimers, corresponding to 500-1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment-protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.     

Line Active Hybrid Lipids Determine Domain Size in Phase Separation of Saturated and Unsaturated Lipids

A simple model of the line activity of a hybrid lipid (e.g., POPC) with one fully saturated chain and one partially unsaturated chain demonstrates that these lipids preferentially pack at curved interfaces between phase-separated saturated and unsaturated domains. We predict that the domain sizes typically range from tens to hundreds of nm, depending on molecular interactions and parameters such as molecular volume and area per headgroup in the bulk fluid phase. The role of cholesterol is taken into account by an effective change in the headgroup areas and the domain sizes are predicted to increase with cholesterol concentration.     

Interaction of heparin with fibronectin and isolated fibronectin domains.

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.     

The positive role of voids in the plasma membrane in growth and energetics of Escherichia coli

Bacterial respiration, endogenous as well as induced respiration by glucose, lactose and glycine betaine, was found to be sensitive to external solute concentration. Permeability of hydrogen peroxide, a non-electrolyte of molecular size between water and urea, through the bacterial membranes changed directly with the rate of respiration (an activity residing in the bacterial plasma membrane) in E. coli and the enhanced permeability and respiratory activity were highly correlated. Hydrogen peroxide permeability and induction of voids (spaces in the matrix of the bilayer into which hydrophobic fluorescent probes partition, which in turn were used to assess the modulation of these cavities) were shown to be a direct and excellent measure of leak conductance. Fluorescence intensity and anisotropy of the extrinsic fluorescent probes (incorporated by growing bacteria in their presence) decreased with increased respiration in bacteria, consistent with lowered molecular restriction and enhanced hydration in the membrane phase for these probes as seen in dimyristoylphosphatidylcholine bilayers due to phase transition. The physical basis of osmotic phenomena, as a relevant (thermodynamic) volume, could relate to water exchange or compression, depending on the osmotic domain. In the domain of compression in bacteria, i.e. well above the isotonic range, the computed activation volume was consistent with voids in the membrane. This study emphasises a major role of leak conductance in bacterial physiology and growth.     

Characterization of high-order diphtheria toxin oligomers

In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.     

重组泛素连接酶PTB-U-box/RING的构建与表达

目的 构建重组泛素连接酶PTB-U-box、PTB-RING,并克隆进入pFLAG-CMV4真核表达载体,为研究靶向降解受体型酪氨酸激酶IGF-IR,抑制肿瘤细胞生长提供基础.方法 分别设计引物,扩增接头分子IRS-1的PTB结构域以及E3泛素连接酶CHIP的U-box、Cbl的RING结构域,再利用重组PCR,将PTB分别与U-box、RING进行融合,双酶切之后将其插入真核表达载体pFLAG-CMV4,经过酶切鉴定及测序后,转染HeLa细胞,Western 印迹验证重组质粒的表达.结果 PCR结果显示PTB-U-box条带大小840 bp,PTB-RING大小为620 bp,重组质粒经酶切鉴定和测序结果正确,转染后可见融合蛋白的表达.结论 成功构建真核重组表达载体pFLAG-CMV4-PTB-U-box和 pFLAG-CMV4-PTB-RING,并且转染HeLa细胞后证实其能够正确表达.