Statistical-mechanical theory of DNA looping

The lack of a rigorous analytical theory for DNA looping has caused many DNA-loop-mediated phenomena to be interpreted using theories describing the related process of DNA cyclization. However, distinctions in the mechanics of DNA looping versus cyclization can have profound quantitative effects on the thermodynamics of loop closure. We have extended a statistical mechanical theory recently developed for DNA cyclization to model DNA looping, taking into account protein flexibility. Notwithstanding the underlying theoretical similarity, we find that the topological constraint of loop closure leads to the coexistence of multiple classes of loops mediated by the same protein structure. These loop topologies are characterized by dramatic differences in twist and writhe; because of the strong coupling of twist and writhe within a loop, DNA looping can exhibit a complex overall helical dependence in terms of amplitude, phase, and deviations from uniform helical periodicity. Moreover, the DNA-length dependence of optimal looping efficiency depends on protein elasticity, protein geometry, and the presence of intrinsic DNA bends. We derive a rigorous theory of loop formation that connects global mechanical and geometric properties of both DNA and protein and demonstrates the importance of protein flexibility in loop-mediated protein-DNA interactions.     

Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha

Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.     

Combination of integrin siRNA and irradiation for breast cancer therapy

Up-regulation of integrin alpha(v)beta(3) has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin alpha(v)beta(3) in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin alpha(v) siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin alpha(v)beta(3)-positive). Integrin alpha(v)beta(3)-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin alpha(v)beta(3) expression in MDA-MB-435 cells and integrin alpha(v) siRNA can effectively reduce both alpha(v) and alpha(v)beta(3) integrin expression, leading to increased radiosensitivity. Integrin alpha(v) siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin alpha(v)beta(3)-positive cancer types.     

Synthesis, characterization and thermal sensitivity of chitosan-based graft copolymers

Novel chitosan-based graft copolymers (CECTS-g-PDMA) were synthesized through homogeneous graft copolymerization of (N,N-dimethylamino)ethyl methacrylate (DMA) onto N-carboxyethylchitosan (CECTS) in aqueous solution by using ammonium persulfate (APS) as the initiator. The effect of polymerization variables, including initiator concentration, monomer concentration, reaction time and temperature, on grafting percentage was studied. XRD, FTIR, DSC and TGA were used to characterize the graft copolymers. Surface-tension measurements, turbidity measurements and temperature-variable (1)H NMR analysis were combined to investigate the thermal sensitivity of CECTS-g-PDMAs in aqueous solution.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae

Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.     

High-performance liquid chromatography coupled with tandem mass spectrometry applied for metabolic study of ginsenoside Rb1 on rat

Liquid chromatography coupled with mass spectrometry and tandem mass spectrometry has been applied to investigate the in vivo metabolism of ginsenoside Rb(1) in rat. Both positive electrospray ionization mass spectrometry and negative electrospray ionization mass spectrometry were used to identify the Rb(1) and its metabolites in rat plasma, urine, and feces samples. Oxygenation and deglycosylation were found to be the major metabolic pathways of Rb(1) in rat. A total of nine metabolites were detected in urine and feces samples collected after intravenous and oral administration. Deglycosylated metabolism of Rb(1) generated other ginsenosides as the major metabolites, such as Rd, Rg(3) or F(2), Rh(2), or C-K. This result indicates that the ginsenoside Rb(1) has many pharmacological activities and could be used as a prodrug.     

Variation between freshwater and terrestrial fungal communities on decaying bamboo culms

Fungal communities on decaying culms of a bamboo host (Phyllostachys bambusoides) from freshwater and adjacent terrestrial habitats were identified. Collections were made at Xiao Bai Long Mountain, Yiliang, Yunnan, China in the winter and summer. In each collection, 100 similar-sized bamboo culms were collected, comprising 50 submerged samples from a stream and 50 terrestrial samples from adjacent riparian vegetation. A total of 82 fungal taxa were recorded from the samples, including 30 ascomycetes and 52 anamorphic fungi. The frequency of occurrence of these fungi were recorded and the Shannon–Weiner indices (H′) were applied to evaluate fungal diversity. The results showed that variation of the fungal diversity between the summer and winter collections was insignificant (0.2<p<0.5). Fungal diversity on submerged bamboo however, was significantly higher than that on terrestrial bamboo (p<0.001). Further findings were that: (1) some commonly recorded freshwater and terrestrial taxa were found in both habitats, but overall there were only 15 overlapping species between the two habitats; (2) the dominant species in each habitat were considerably different, and (3) only a few fungi were dominant, while most species were rare, being recorded only once or twice. Factors responsible for the distribution patterns and variations in composition of the fungal communities are discussed.     

Prediction of protease types in a hybridization space

Regulating most physiological processes by controlling the activation, synthesis, and turnover of proteins, proteases play pivotal regulatory roles in conception, birth, digestion, growth, maturation, ageing, and death of all organisms. Different types of proteases have different functions and biological processes. Therefore, it is important for both basic research and drug discovery to consider the following two problems. (1) Given the sequence of a protein, can we identify whether it is a protease or non-protease? (2) If it is, what protease type does it belong to? Although the two problems can be solved by various experimental means, it is both time-consuming and costly to do so. The avalanche of protein sequences generated in the post-genetic era has challenged us to develop an automated method for making a fast and reliable identification. By hybridizing the functional domain composition and pseudo-amino acid composition, we have introduced a new method called "FunD-PseAA predictor" that is operated in a hybridization space. To avoid redundancy and bias, demonstrations were performed on a dataset where none of the proteins has >or=25% sequence identity to any other. The overall success rate thus obtained by the jackknife cross-validation test in identifying protease and non-protease was 92.95%, and that in identifying the protease type was 94.75% among the following six types: (1) aspartic, (2) cysteine, (3) glutamic, (4) metallo, (5) serine, and (6) threonine. Demonstration was also made on an independent dataset, and the corresponding overall success rates were 98.36% and 97.11%, respectively, suggesting the FunD-PseAA predictor is very powerful and may become a useful tool in bioinformatics and proteomics.     

Binding of Src to Na+/K+-ATPase forms a functional signaling complex

We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.