Diet composition and feeding ecology of the naked goby Gobiosoma bosc (Gobiidae) from four western Atlantic estuaries.

The feeding ecology of the small-bodied benthic naked goby Gobiosoma bosc, a western Atlantic species that occurs in estuaries and other inshore habitats from Connecticut to Texas U.S.A., was investigated in a total of four estuaries spanning South Carolina, North Carolina, Maryland and New Jersey. Gut content analysis of 391 individuals revealed that G. bosc is a benthic microcarnivore that feeds primarily on polychaetes, gammarid amphipods and harpacticoid copepods. Diet composition varied with body size, tidal creek within an estuary and geographic region. Analyses of gut fullness suggest that G. bosc is a daytime visual predator and that nest and egg guarding during the reproductive season reduce foraging activity in mature males. Additionally, G. bosc infected with adult digenean parasites of the gut foraged more intensely than uninfected individuals, a relationship that was strongest for reproductively mature males. Regionally, significant variation in dietary breadth was documented and may reflect a foraging response to a decrease in prey diversity moving from estuaries of higher salinity and lower latitude to estuaries of lower salinity and higher latitude. These results contribute to an understanding of the life history of G. bosc and the role played by this common species in estuarine food webs.

Genetic population structure of spotted seatrout Cynoscion nebulosus along the south-eastern U.S.A.

Analyses of the genetic population structure of spotted seatrout Cynoscion nebulosus along the south-eastern U.S. coast using 13 microsatellites suggest significant population differentiation between fish in North Carolina (NC) compared with South Carolina (SC) and Georgia (GA), with New River, NC, serving as an area of integration between northern and southern C. nebulosus. Although there is a significant break in gene flow between these areas, the overall pattern throughout the sampling range represents a gradient in genetic diversification with the degree of geographic separation. Latitudinal distance and estuarine density appear to be main drivers in the genetic differentiation of C. nebulosus along the south-eastern U.S. coast. The isolation-by-distance gene-flow pattern creates fine-scale differences in the genetic composition of proximal estuaries and dictates that stocking must be confined to within 100 km of the location of broodstock collection in order to maintain the natural gradient of genetic variation along the south-eastern U.S. coast.

Molecular modeling of the amino-terminal zinc ring domain of BRCA1.

The equine herpes virus zinc ring domain nuclear magnetic resonance structure was used for homology-based modeling of the amino-terminal zinc ring domain of the BRCA1 breast and ovarian cancer susceptibility gene. The zinc ring domain of BRCA1 is of particular interest because it is the location of significant and frequently occurring missense (Cys(61)Gly, Cys(64)Gly, and Cys(64)Tyr) and frameshift (185delAG) mutations observed in several high-risk kindreds. The BRCA1 zinc ring domain possesses 54% sequence similarity with the equine herpes virus zinc ring domain. The model structure undergoes little conformational variance after 140 ps of solvated molecular dynamics. This model proposes BRCA1 zinc ring domain residues that may play a role in DNA binding and/or protein-protein interactions. These predictions provide a point of departure for the design of mutants to probe BRCA1 zinc ring domain functionality.

p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay.

Changes in promoter specificity and binding affinity that may be associated with p53 mutations or post-translational modifications are useful in understanding p53 structure/function relationships and categorizing tumor mutations. We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and affinity. The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied. p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identified at low level of p53 protein expression. All the p53 mutants obtained with this screen were located in the DNA binding domain. Two out of 17 supertrans mutants have been found in tumors. Six mutations were in the L1 loop region between amino acids 115 and 124. The transactivation potential of a panel of supertrans p53 mutants on different promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some activity with all promoters, we found different patterns of induction based on strength and promoter specificity. In particular none of the mutants was supertrans for the p21 responsive element. Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53. Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters. These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding specific interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols. The approach may also prove useful in identifying p53 tumor mutations.

The coalescent process in models with selection.

Statistical properties of the process describing the genealogical history of a random sample of genes are obtained for a class of population genetics models with selection. For models with selection, in contrast to models without selection, the distribution of this process, the coalescent process, depends on the distribution of the frequencies of alleles in the ancestral generations. If the ancestral frequency process can be approximated by a diffusion, then the mean and the variance of the number of segregating sites due to selectively neutral mutations in random samples can be numerically calculated. The calculations are greatly simplified if the frequencies of the alleles are tightly regulated. If the mutation rates between alleles maintained by balancing selection are low, then the number of selectively neutral segregating sites in a random sample of genes is expected to substantially exceed the number predicted under a neutral model.

Evolution and extinction of transposable elements in Mendelian populations.

A model of the evolution of a transposable element family in a Mendelian host population is proposed that incorporates heritable phenotypic mutations in the elements. The temporal behavior of the numbers of mutant and wild-type elements is studied, and the expected extinction time of the transposable element family is examined. Our results indicate that, if the mutant can be transposed equally well in the presence of the wild type, then it can be expected to be found in preponderance, whereas elements, such as retroviruses, where the transposing genome and its phenotypic expression are coupled, may be characterized by a low mutant frequency.

Homology modeling and molecular dynamics simulation of human prothrombin fragment 1.

The crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hf1/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long-range ionic forces was essential to reach a stable solution structure. The gamma-carboxyglutamic acid (Gla) domain and the kringle domain of hf1/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K-dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.

Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6-pentakisphosphate 3-phosphatase activity.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375-13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P(5) (K(m)=7.1 microM) and PtdIns(3,4,5)P(3) (K(m)=26 microM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P(5) levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P(5) to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

An all-atom solution-equilibrated model for human extrinsic blood coagulation complex (sTF-VIIa-Xa): a protein-protein docking and molecular dynamics refinement study.

Tissue factor (TF)-bound factor (F)VIIa plays a critical role in activating FX, an event that rapidly results in blood coagulation. Despite recent advances in the structural information about soluble TF (sTF)-bound VIIa and Xa individually, the atomic details of the ternary complex are not known. As part of our long-term goal to provide a structural understanding of the extrinsic blood coagulation pathway, we built an all atom solution-equilibrated model of the human sTF-VIIa-Xa ternary complex using protein-protein docking and molecular dynamics (MD) simulations. The starting structural coordinates of sTF-VIIa and Xa were derived from dynamically equilibrated solution structures. Due to the flexible nature of the light-chain of the Xa molecule, a three-stage docking approach was employed in which SP (Arg195-Lys448)/EGF2 (Arg86-Arg139), EGF1 (Asp46-Thr85) and GLA (Ala1-Lys45) domains were docked in a sequential manner. The rigid-body docking approach of the FTDOCK method in conjunction with filtering based on biochemical knowledge from experimental site-specific mutagenesis studies provided the strategy. The best complex obtained from the docking experiments was further refined using MD simulations for 3 ns in explicit water. In addition to explaining most of the known experimental site-specific mutagenesis data pertaining to sTF-VIIa, our model also characterizes likely enzyme-binding exosites on FVIIa and Xa that may be involved in the ternary complex formation. According to the equilibrated model, the 140s loop of VIIa serves as the key recognition motif for complex formation. Stable interactions occur between the FVIIa 140s loop and the FXa -strand B2 region near the sodium-binding domain, the 160 s loop and the N-terminal activation loop regions. The helical-hydrophobic stack region that connects the GLA and EGF1 domains of VIIa and Xa appears to play a potential role in the membrane binding region of the ternary complex. The proposed model may serve as a reasonable structural basis for understanding the exosite-mediated substrate recognition of sTF-VIIa and to advance understanding of the TFPI-mediated regulatory pathway of the extrinsic blood coagulation cascade.

The roles of individual amino acids in altering substrate specificity of the P450 2a4/2a5 enzymes.

A single amino acid substitution is sufficient to alter substrate specificity of P450 enzymes. Mouse P450 2a5, for example, has its substrate specificity converted from coumarin 7- to testosterone 15 alpha-hydroxylase activity by the substitution of Phe at position 209 to Leu. Furthermore, placing Asn at this position confers a novel corticosterone 15 alpha-hydroxylase activity to this P450. Recent site-directed mutational studies show the presence of the topologically common residues, each of which can determine the specificities of various mammalian P450s. For instance, residue 209 (in 2a5) corresponds to a residue at position 206 in rat P4502B1 that regulates its steroid hydroxylase activity. High substrate specificity often observed in an individual P450, therefore, can be determined and altered by the identities of a few critical residues. The structural flexibility of the substrate-heme pocket may also provide P450 enzymes with the ability to display a broad range of substrate specificities. Understanding the underlying principles whereby the flexible pocket determines P450 activities may lead us to the prediction of P450 activities based on the identities of key amino acid residues.

Requirement for G proteins in insulin-like growth factor-I-induced growth of prostate cells.

Elevated levels of IGF-I in the circulation are associated with increased risk for the development of prostate cancer in men, and transgenic expression of human IGF-I in mouse epithelial prostate cells results in spontaneous prostate tumorigenesis. Little, however, is known about the mechanisms involved in the IGF-I-regulated growth of prostate cells. Here, we have demonstrated that treatment with IGF-I induces the activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and the growth of human prostate cells. Stimulation with IGF-I also promoted the tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Signal relay from IGF-I to ERK requires heterotrimeric G proteins and EGFR; inhibition of Gi/o protein activation by pertussis toxin, or EGFR by tyrphostin AG1478 obliterated the ability of IGF-I to promote ERK activation. Further, treatment with pertussis toxin inhibited the IGF-I-mediated prostate cell growth. These data demonstrated the requirement of heterotrimeric G proteins in IGF-I-regulated prostate cell growth and suggest the potential utility of the G proteins as effective drug targets to combat this common cancer.

A theoretical study of the binding of polychlorinated biphenyls (PCBs), dibenzodioxins, and dibenzofuran to human plasma prealbumin.

Binding energies to human plasma prealbumin using the energy minimization program AMBER are found for a series of polychlorinated biphenyls, dibenzodioxins, and dibenzofuran. Corrections for solvation free energies of the chlorinated analogues lead to estimates of the differential free energies of complex formation. These are compared in a number of cases to known experimental log (KPCB/Kref) values. The theory correctly separates strong, intermediate, and nonbinders. On the basis of calculations, 2,3,7,8-tetrachlorodibenzodioxin and 2,3,7,8-tetrachlorodibenzofuran are predicted to be strong binders, 3,3',5,5'-tetrachlorodiphenoquinone is predicted to be a weak binder, and octachlorodibenzodioxin is predicted to not bind at all. This theoretical model for prealbumin interactions may be of use in estimating the toxic potential of PCBs and related halogenated aromatic hydrocarbons of environmental importance.

Modeling human zymogen factor IX.

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.

Selected new developments in computational chemistry.

Molecular dynamics is a general technique for simulating the time-dependent properties of molecules and their environments. Quantum mechanics, as applied to molecules or clusters of molecules, provides a prescription for predicting properties exactly (in principle). It is reasonable to expect that both will have a profound effect on our understanding of environmental chemistry in the future. In this review, we consider several recent advances and applications in computational chemistry.

Molecular modeling studies suggest that zinc ions inhibit HIV-1 protease by binding at catalytic aspartates.

Human immunodeficiency virus type 1 protease is inhibited in vitro by zinc ions at neutral pH. The binding site of these ions is not known; however, experimental data suggest that binding may occur in the active site. To examine the possibility of zinc binding in the active site, molecular dynamics simulations in the presence and absence of zinc have been carried out to 200 psec. The results are compared with the 2.8-A crystallographic structures of a synthetic HIV-1 protease, and a zinc binding site at the catalytic aspartate residues (Asp-25, Asp-25') is proposed. Molecular dynamics simulations show that the zinc ion remains stably bound in this region, coordinating the carboxylate side chains of both aspartate residues. Interaction with zinc does not disrupt the dimeric structure of the protein or significantly alter the structure of the active site. These data are consistent with experimental studies of HIV-1 protease inhibition by zinc and give strong evidence that this is the binding site that leads to inactivation.

Structural flexibility and functional versatility of cytochrome P450 and rapid evolution.

P450 represents a large group of heme-thiolate enzymes that exhibit remarkably diverse activities for the metabolism of numerous endogenous and exogenous chemicals. Recent site-directed mutagenesis studies indicate that a single mutation at any of the key residues can be enough to alter the substrate and/or product specificities in the P450 activities. Molecular modeling predicts that these key residues are located within the substrate heme pocket. Structural elements involved in diversifying P450 activity appear to correspond to the B' helix, the F helix and the F/G interhelical loop in the bacterial P450s. Structures represented by these regions are extremely variable despite the fact that the core of the P450 substrate pocket is well conserved. A mutation within these regions may result in a significant geometrical alteration of the pocket and lead to diversify the P450 activity. Phylogenetical analysis shows a relatively high rate of nonsynonymous substitution within these substrate binding regions. The functional versatility of P450 can thus be largely accounted for in terms of pocket change brought about by rapid mutations.

Theoretical and experimental measures of DNA helix stability and their relation to sequence specific repair of O6-ethylguanine lesions.

Recent work (Breslauer et al. (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 3746) has provided a method for calculating empirical thermodynamic quantities for helix to coil transitions from the base sequence of any oligomer. It is shown in this work that the DNA helix binding energy, calculated with the AMBER force field, for 9-mers of the type 5'-GGGXGeYGGG-3', where X and Y are any base and the central Ge is O6-ethylguanine, correlates well with the empirical delta G for helix to strand transitions. The mutation spectrum of ethane methylsulfonate (EMS) in the lacI gene of Escherichia coli can be modeled using the calculated local binding energy but the empirical free energies, enthalpies and melting temperatures predict these levels of repair less well. The relation of the binding energy to the mutation spectrum can be somewhat improved by including entropic effects in a theoretical free energy of binding as given by delta G theoretical identical to delta E binding - T delta S.

Study of the docking of competitive inhibitors at a model of tyrosinase active site: insights from joint broken-symmetry/Spin-Flip DFT computations and ELF topological analysis.

Following our previous study (Piquemal et al., New J. Chem., 2003, 27, 909), we present here a DFT study of the inhibition of the Tyrosinase enzyme. Broken-symmetry DFT computations are supplemented with Spin-Flip TD-DFT calculations, which, for the first time, are applied to such a dicopper enzyme. The chosen biomimetic model encompasses a dioxygen molecule, two Cu(II) cations, and six imidazole rings. The docking energy of a natural substrate, namely phenolate, together with those of several inhibitor and non-inhibitor compounds, are reported and show the ability of the model to rank the most potent inhibitors in agreement with experimental data. With respect to broken-symmetry calculations, the Spin-Flip TD-DFT approach reinforces the possibility for theory to point out potent inhibitors: the need for the deprotonation of the substrates, natural or inhibitors, is now clearly established. Moreover, Electron Localization Function (ELF) topological analysis computations are used to deeply track the particular electronic distribution of the Cu-O-Cu three-center bonds involved in the enzymatic Cu(2)O(2) metallic core (Piquemal and Pilmé, J. Mol. Struct.: Theochem, 2006, 77, 764). It is shown that such bonds exhibit very resilient out-of-plane density expansions that play a key role in docking interactions: their 3D-orientation could be the topological electronic signature of oxygen activation within such systems.