Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercatorum

rDNA nontranscribed spacer (NTS) lengths of Drosophila mercatorum have been measured in individuals from several geographic regions. Individuals from the different geographic subpopulations share some length fragments but are in general distinct. The length differences, both within and between individuals, arise from different copy numbers of a 250-bp repeating unit that is localized to one part of the NTS. In addition to the length differences caused by the 250-bp repeat, there is a Y chromosome (male)-specific length variant elsewhere in the NTS that is approximately 70 bp shorter than the NTS fragment from the X chromosome. Sexual dimorphism seems to be present in all Drosophila. Also, D. mercatorum has fewer NTS length variants per individual than does D. melanogaster while possessing comparable levels of restriction- site polymorphism. The mechanisms that may cause this pattern of variation are selection, gene conversion, and unequal recombination.      

Anticancer activity profiling of parthenolide analogs generated via P450-mediated chemoenzymatic synthesis

The plant-derived sesquiterpene lactone parthenolide (PTL) was recently found to possess promising anticancer activity but elaboration of this natural product scaffold for optimization of its pharmacological properties has proven challenging via available chemical methods. In this work, P450-catalyzed C–H hydroxylation of positions C9 and C14 in PTL was coupled to carbamoylation chemistry to yield a panel of novel carbamate-based PTL analogs (‘parthenologs’). These compounds, along with a series of other C9- and C14-functionalized parthenologs obtained via O–H acylation, alkylation, and metal-catalyzed carbene insertion, were profiled for their cytotoxicity against a diverse panel of human cancer cell lines. These studies led to the discovery of several parthenologs with significantly improved anticancer activity (2–14-fold) compared to the parent molecule. Most interestingly, two PTL analogs with high cytotoxicity (LC₅₀ ～ 1–3 μM) against T cell leukemia (Jurkat), mantle cell lymphoma (JeKo-1), and adenocarcinoma (HeLa) cells as well as a carbamate derivative with potent activity (LC₅₀ = 0.6 μM) against neuroblastoma cells (SK-N-MC) were obtained. In addition, these analyses resulted in the identification of parthenologs featuring both a broad spectrum and tumor cell-specific anticancer activity profile, thus providing valuable probes for the future investigation of biomolecular targets that can affect cell viability across multiple as well as specific types of human cancers. Altogether, these results highlight the potential of P450-mediated chemoenzymatic C–H functionalization toward tuning and improving the anticancer activity of the natural product parthenolide.     

The influence of optimization target selection on the structure of arterial tree models generated by constrained constructive optimization

The computational method of constrained constructive optimization was used to generate complex arterial model trees by optimization with respect to a target function. Changing the target function also changes the tree structure obtained. For a parameterized family of target functions a series of trees was created, showing visually striking differences in structure that can also be quantified by appropriately chosen numerical indexes. Blood transport path length, pressure profile, and an index for relative segment orientation show clear dependencies on the optimization target, and the nature of changes can be explained on theoretical grounds. The main goal was to display, quantify, and explain the structural changes induced by different optimization target functions.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

The role of marine reserves in the replenishment of a locally impacted population of anemonefish on the Great Barrier Reef

The development of parentage analysis to track the dispersal of juvenile offspring has given us unprecedented insight into the population dynamics of coral reef fishes. These tools now have the potential to inform fisheries management and species conservation, particularly for small fragmented populations under threat from exploitation and disturbance. In this study, we resolve patterns of larval dispersal for a population of the anemonefish Amphiprion melanopus in the Keppel Islands (southern Great Barrier Reef). Habitat loss and fishing appear to have impacted this population and a network of no‐take marine reserves currently protects 75% of the potential breeders. Using parentage analysis, we estimate that 21% of recruitment in the island group was generated locally and that breeding adults living in reserves were responsible for 79% (31 of 39) of these of locally produced juveniles. Overall, the network of reserves was fully connected via larval dispersal; however, one reserve was identified as a critical source of larvae for the island group. The population in the Keppel Islands also appears to be well‐connected to other source populations at least 60 km away, given that 79% (145 of 184) of the juveniles sampled remained unassigned in the parentage analysis. We estimated the effective size of the A. melanopus metapopulation to be 745 (582–993 95% CI) and recommend continued monitoring of its genetic status. Maintaining connectivity with populations beyond the Keppel Islands and recovery of local recruitment habitat, potentially through active restoration of host anemone populations, will be important for its long‐term persistence.     

Anemonefish depletion reduces survival,growth, reproduction and fishery productivity of mutualistic anemone–anemonefish colonies

Intimate knowledge of both partners in a mutualism is necessary to understand the ecology and evolution of each partner, and to manage human impacts that asymmetrically affect one of the partners. Although anemonefishes and their host anemones are iconic mutualists and widely sought by ornamental fisheries, the degree to which anemones depend on anemonefishes, and thus the colony-level effects of collecting anemonefishes, is not well understood. We tracked the size and abundance of anemone Entacmaea quadricolor and anemonefish Amphiprion melanopus colonies for 3 yr after none, some, or all of the resident anemonefish were experimentally removed. Total and partial removal of anemonefish had rapid and sustained negative effects on growth, reproduction and survival of anemones, as well as cascading effects on recruitment and productivity of anemonefish in the remaining colony. As predicted, total removal of anemonefish caused acute declines in size and abundance of anemones, although most anemone colonies (76 %) slowly resumed growth and reproduction after the arrival of anemonefish recruits, which subsequently grew and defended the hosts. Partial removal of anemonefish had similar but typically less severe effects on anemones. Remarkably, the colony-level effects on anemones and anemonefish were proportional to the size and number of anemonefish that were experimentally removed. In particular, anemone survival and anemonefish productivity were highest when one or more adult anemonefish remained in the colony, suggesting that adult fish not only enhanced the protection of anemones, but also increased the recruitment and/or survival of conspecifics. We conclude that the relationship between E. quadricolor and A. melanopus is not only obligate, but also demographically rigid and easily perturbed by anemonefish fisheries. Clearly, these two species must be managed together as a unit and with utmost precaution. To this end, we propose several tangible management actions that will help to minimize fishing effects.     

Relaxation effects in the gel electrophoresis of DNA in intermittent fields

The electrophoretic mobility of restriction fragments of lambda DNA in agarose gels declines if the field is intermittent rather than continuous, with a greater effect on the longer fragments. The changes are compatible with the assumption of two exponential relaxation processes for field-dependent configurational changes, one when the field is turned on and another when it terminates. The length dependence at the extrapolated limit of mobility for short pulses with long intervals corresponds closely to the simple inverse proportionality to length expected from theoretical considerations when the molecular configuration is not affected by the electric field. Simple intermittent fields would allow separation of longer molecules than can ordinarily be resolved. The relaxation times for both the change in conformation imposed by the field and the return to field-free conformation vary as approximately the second power of the length of the molecule, independent of the salt concentration or field strength and varying only slightly with gel density. These relations are not in good agreement with properties expected from reputation theory, and they suggest that a different mechanism must be invoked for the electrophoretic migration of long DNA molecules at ordinary values of field strength.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.