Using ecological networks to answer questions in global biogeography and ecology

Ecological networks have classically been studied at site and landscape scales, yet recent efforts have been made to collate these data into global repositories. This offers an opportunity to integrate and upscale knowledge about ecological interactions from local to global scales to gain enhanced insights from the mechanistic information provided by these data. By drawing on existing research investigating patterns in ecological interactions at continental to global scales, we show how data on ecological networks, collected at appropriate scales, can be used to generate an improved understanding of many aspects of ecology and biogeography—for example, species distribution modelling, restoration ecology and conservation. We argue that by understanding the patterns in the structure and function of ecological networks across scales, it is possible to enhance our understanding of the natural world.     

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P1) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state (P⁺I^?) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P⁺ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P⁺I^? (or P^F) lives approx. 15 ns. It decays, in part, into a longer lived state (P^R), which appears to be a triplet state. State P^R decays with an exponential time of approx. 55 μs. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P1. Most of P1 then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P1 or P⁺ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I^? suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.     

Comparative genomics as a tool for gene discovery

With the increasing availability of data from multiple eukaryotic genome sequencing projects, attention has focused on interspecific comparisons to discover novel genes and transcribed genomic sequences. Generally, these extrinsic strategies combine ab initio gene prediction with expression and/or homology data to identify conserved gene candidates between two or more genomes. Interspecific sequence analyses have proven invaluable for the improvement of existing annotations, automation of annotation, and identification of novel coding regions and splice variants. Further, comparative genomic approaches hold the promise of improved prediction of terminal or small exons, microRNA precursors, and small peptide-encoding open reading frames--sequence elements that are difficult to identify through purely intrinsic methodologies in the absence of experimental data.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X-ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6₅22 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent. © 1996 Wiley-Liss, Inc.     

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

Circular zymogens of human ribonuclease 1

The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 10⁴‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.     

A Fish Eye Out of Water: Ten Visual Opsins in the Four-Eyed Fish,Anableps anableps

The “four-eyed” fish Anableps anableps has numerous morphological adaptations that enable above and below-water vision. Here, as the first step in our efforts to identify molecular adaptations for aerial and aquatic vision in this species, we describe the A. anableps visual opsin repertoire. We used PCR, cloning, and sequencing to survey cDNA using unique primers designed to amplify eight sequences from five visual opsin gene subfamilies, SWS1, SWS2, RH1, RH2, and LWS. We also used Southern blotting to count opsin loci in genomic DNA digested with EcoR1 and BamH1. Phylogenetic analyses confirmed the identity of all opsin sequences and allowed us to map gene duplication and divergence events onto a tree of teleost fish. Each of the gene-specific primer sets produced an amplicon from cDNA, indicating that A. anableps possessed and expressed at least eight opsin genes. A second PCR-based survey of genomic and cDNA uncovered two additional LWS genes. Thus, A. anableps has at least ten visual opsins and all but one were expressed in the eyes of the single adult surveyed. Among these ten visual opsins, two have key site haplotypes not found in other fish. Of particular interest is the A. anableps-specific opsin in the LWS subfamily, S180γ, with a SHYAA five key site haplotype. Although A. anableps has a visual opsin gene repertoire similar to that found in other fishes in the suborder Cyprinodontoidei, the LWS opsin subfamily has two loci not found in close relatives, including one with a key site haplotype not found in any other fish species. A. anableps opsin sequence data will be used to design in situ probes allowing us to test the hypothesis that opsin gene expression differs in the distinct ventral and dorsal retinas found in this species.