Phylogenetic analyses among octocorals (Cnidaria): mitochondrial and nuclear DNA sequences (lsu-rRNA, 16S and ssu-rRNA, 18S) support two convergent clades of branching gorgonians

Gorgonian octocorals lack corroborated hypotheses of phylogeny. This study reconstructs genealogical relationships among some octocoral species based on published DNA sequences from the large ribosomal subunit of the mitochondrial RNA (lsu-rRNA, 16S: 524bp and 21 species) and the small subunit of the nuclear RNA (ssu-rRNA, 18S: 1815bp and 13 spp) using information from insertions-deletions (INDELS) and the predicted secondary structure of the lsu-rRNA (16S). There were seven short (3-10bp) INDELS in the 18S with consistent phylogenetic information. The INDELS in the 16S corresponded to informative signature sequences homologous to the G13 helix found in Escherichia coli. We found two main groups of gorgonian octocorals using a maximum parsimony analysis of the two genes. One group corresponds to deep-water taxa including species from the suborders Calcaxonia and Scleraxonia characterized by an enlargement of the G13 helix. The second group has species from Alcyoniina, Holaxonia and again Scleraxonia characterized by insertions in the 18S. Gorgonian corals, branching colonies with a gorgonin-containing flexible multilayered axis (Holaxonia and Calcaxonia), do not form a monophyletic group. These corroborated results from maternally inherited (16S) and biparentally inherited (18S) genes support a hypothesis of independent evolution of branching in the two octocoral clades.     

Insights into antifolate resistance from malarial DHFR-TS structures

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.     

A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

Directly observed therapy (DOT) for individuals with HIV: successes and challenges

Many HIV-infected individuals have not reaped the benefits of combination antiretroviral therapy due to inability either to adhere to medications or to access care. It is now recognized that innovative approaches are needed to increase access and adherence to highly active antiretroviral therapy (HAART), especially among these hard-to-reach populations. Due to the success of directly observed therapy (DOT) for the treatment of Mycobacterium tuberculosis (TB), our group and others have questioned whether DOT can be adapted to deliver HAART to hard-to-reach communities. In this review, we discuss the results of pilot programs that have utilized DOT in multiple different settings and use case studies to explore the diverse issues that can arise when implementing these programs. As we continue to gain more experience with observed therapy, we will be able to better identify the key components for a successful intervention.     

Pigment dynamics and autumn leaf senescence in a New England deciduous forest,eastern USA

The leaves of woody plants at Harvard Forest in Central Massachusetts, USA, changed color during senescence; 70% (62/89) of the woody species examined anatomically contained anthocyanins during senescence. Anthocyanins were not present in summer green leaves, and appeared primarily in the vacuoles of palisade parenchyma cells. Yellow coloration was a result of the unmasking of xanthophyll pigments in senescing chloroplasts. In nine red-senescing species, anthocyanins were not detectable in mature leaves, and were synthesized de novo in senescence, with less than 20µg cm^–2 of chlorophyll remaining. Xanthophyll concentrations declined in relation to chlorophyll to the same extent in both yellow- and red-leaved taxa. Declines in the maximum photosystemII quantum yield of leaves collected prior to dawn were only slightly less in the red-senescing species, indicating no long-term protective activity. Red-leaved species had significantly greater mass/area and lower chlorophylla/b ratios during senescence. Nitrogen tissue concentrations in mature and senescent leaves negatively correlated to anthocyanin concentrations in senescent leaves, weak evidence for more efficient nitrogen resorption in anthocyanic species. Shading retarded both chlorophyll loss and anthocyanin production in Cornus alternifolia, Acer rubrum, Acer saccharum, Quercus rubra and Viburnum alnifolium. It promoted chlorophyll loss in yellow-senescing Fagus grandifolia. A reduced red:far-red ratio did not affect this process. Anthocyanins did not increase leaf temperatures in Q.rubra and Vaccinium corymbosum on cold and sunny days. The timing of leaf-fall was remarkably constant from year to year, and the order of senescence of individual species was consistent.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

Effects of pulp mill solids and three composts on early growth of tomatoes

Compost has been proposed as a means of simultaneously diverting organic materials from landfills while producing a valuable product that improves tilth, organic matter content and nutrient supply of agricultural soils. Composts manufactured from different source materials may have markedly different properties however, even if they meet all regulatory requirements. We compared the capacity of composts made from three different combinations of organic wastes (horse manure and bedding, mink farm wastes, municipal solid waste (MSW) and sewage sludge) along with clarifier solids from a chemo-thermomechanical pulp mill, to enhance the growth of tomato (Lycopersicon esculentum L.) seedlings grown in nutrient-poor organic potting soil. Germination and seedling emergence of tomatoes, cress (Lapidium sativum L.) or radish (Raphanus sativus L.) were tested to assess phytotoxicity of the four amendments. Mink farm compost and horse manure compost stimulated root and shoot growth of tomato seedlings but MSW compost and pulp mill solids were strongly inhibitory. MSW compost and unamended potting soil also inhibited seedling emergence and pulp mill solids produced stunting and deformities in radish and cress seedlings. Both toxic constituents and nutrient imbalances may be responsible for the growth-inhibiting effects of these amendments. Application of pulp mill solids to agricultural soil without composting may lead to deleterious effects on vegetable crops.