Effect of bovine manure on fecal coliform attachment to soil and soil particles of different sizes

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.     

Homologous desensitization of signalling by the alpha (alpha) isoform of the human thromboxane A2 receptor: a specific role for nitric oxide signalling

Thromboxane (TX) A(2) plays a central role in hemostasis, regulating platelet activation status and vascular tone. We have recently established that the TP beta isoform of the human TXA(2) receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TP alpha isoform. TP alpha undergoes profound agonist-induced desensitization of signalling (intracellular calcium mobilization and inositol 1,4,5 trisphosphate generation) in response to the TXA(2) mimetic U46619 but, unlike that of TP beta, this is independent of GRKs. Similar to TP beta, TP alpha undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser(145) within intracellular domain (IC)(2) represents the key phospho-target. TP alpha also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr(337) and Ser(331), respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors L-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TP alpha involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser(145) partially and transiently impairs TP alpha signalling while PKG- and PKC-phosphorylation at both Ser(331) and Thr(337), respectively, within its C-tail domain profoundly desensitizes TP alpha, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TP alpha.     

Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Palmitoylation of the TPbeta isoform of the human thromboxane A2 receptor. Modulation of G protein: effector coupling and modes of receptor internalization

Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPalpha and TPbeta isoforms of the human prostanoid thromboxane (TX) A2 receptor (TP) are palmitoylated and to assess the functional consequences thereof. Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPbeta is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPalpha is not palmitoylated. Impairment of palmitoylation did not affect TPbeta expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPbetaC347S and the non-palmitoylated TPbetaC347,373,377S, but not by TPbetaC373S or TPbetaC373,377S, was significantly reduced relative to the wild type TPbeta suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cbeta effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPbeta internalization with TPbetaC373S, TPbetaC373,377S and TPbetaC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPbetaC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPbeta. The deficiency in agonist-induced internalization by TPbetaC347S, but not by TPbetaC373,377 nor TPbeta(C347,373,377S), was overcome by over-expression of either beta-arrestin1 or beta-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPbeta at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the beta-arrestin-dependent agonist-induced pathway or by the beta-arrestin-independent tonic internalization pathway.     

Nanotechnological applications in medicine

Nanotechnology-based tools and techniques are rapidly emerging in the fields of medical imaging and targeted drug delivery. Employing constructs such as dendrimers, liposomes, nanoshells, nanotubes, emulsions and quantum dots, these advances lead toward the concept of personalized medicine and the potential for very early, even pre-symptomatic, diagnoses coupled with highly-effective targeted therapy. Highlighting clinically available and preclinical applications, this review explores the opportunities and issues surrounding nanomedicine.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

Multilocus nuclear sequences reveal intra- and interspecific relationships among chromosomally polymorphic species of cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae, a pair of sibling species endemic to North America, constitute an important model system to study ecological genetics and the evolution of reproductive isolation. This species pair can produce fertile hybrids in some crosses and are sympatric in a large part of their ranges. Despite the potential for hybridization in nature, however, evidence of introgression has not been rigorously sought. Further, the evolutionary relationships within and among the geographically distant populations of the two species have not been characterized in detail using high-resolution molecular studies. Both species have six chromosomes: five large acrocentrics and one 'dot' chromosome. Fixed inversion differences between the species exist in three chromosomes (X, 2 and 3) while three are colinear (4, 5 and 6), suggesting that were introgression to occur, it would be most likely in the colinear chromosomes. We utilized nucleotide sequence variation at multiple loci on five chromosomes to test for evidence of introgression, and to test various scenarios for the evolutionary relationships of these two species and their populations. While we do not find evidence of recent introgression, loci in the colinear chromosomes appear to have participated in exchange in the past. We also found considerable population structure within both species. The level of differentiation discovered among D. arizonae populations was unexpectedly high and suggests that its populations, as well as those of D. mojavensis, may be themselves undergoing incipient speciation and merit further attention.     

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.     

Effect of Bovine Manure on Fecal Coliform Attachment to Soil and Soil Particles of Different Sizes

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.