Molecular cytogenetic assignment of genes to bovine chromosome 5

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines.     

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.     

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.     

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.     

Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate

Resistance to anticancer drugs has been attributed to an array of cellular changes. The multidrug resistance-related protein (MRP1) is an efflux pump whose overexpression confers resistance to several classes of drugs, such as the anthracyclines, epipodophyllotoxins, and vinca alkaloids. These drugs are mainstays in cancer therapy. MRP1 overexpression is hypothesized to be a causative agent of clinical treatment failure. Consistently accurate methods for detecting this protein are necessary to further understand its biology and delineate its possible clinical relevance. Flow cytometric analysis of multidrug resistance (MDR) is a valuable method to evaluate both antigen expression and function. Using flow cytometry, we assayed MRP1 functional activity in pediatric leukemic blasts and an array of MDR+ and WT cell lines. We conclude that calcein AM, when used in a retention assay with MRP1-specific modulators, is able to reliably detect MRP functional activity. 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF AM) transport is not indicative of MRP1 overexpression. .