Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.     

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that β2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21?days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4?mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9?days of treatment, while hypertrophy was observed only in EDL after 9?days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14?days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.     

QTL analyses for seed iron and zinc concentrations in an intra-genepool population of Andean common beans (Phaseolus vulgaris L.)

Legumes provide essential micronutrients that are found only in low amounts in the cereals or root crops. An ongoing project at CIAT has shown that the legume common bean is variable in the amount of seed minerals (iron, zinc, and other elements), vitamins, and sulfur amino acids that they contain and that these traits are likely to be inherited quantitatively. In this study we analyzed iron and zinc concentrations in an Andean recombinant inbred line (RIL) population of 100 lines derived from a cross between G21242, a Colombian cream-mottled climbing bean with high seed iron/zinc and G21078, an Argentinean cream seeded climbing bean with low seed iron/zinc. The population was planted across three environments; seed from each genotype was analyzed with two analytical methods, and quantitative trait loci (QTL) were detected using composite interval mapping and single-point analyses. A complete genetic map was created for the cross using a total of 74 microsatellite markers to anchor the map to previously published reference maps and 42 RAPD markers. In total, nine seed mineral QTL were identified on five linkage groups (LGs) with the most important being new loci on b02 and other QTL on b06, b08, and b07 near phaseolin. Seed weight QTL were associated with these on b02 and b08. These Andean-derived QTL are candidates for marker-assisted selection either in combination with QTL from the Mesoamerican genepool or with other QTL found in inter and intra-genepool crosses, and the genetic map can be used to anchor other intra-genepool studies.     

Contrasting community compensatory trends in alternative successional pathways in central Amazonia

Based on eight years of annual censuses in secondary forests in central Amazonia, we compared successional dynamics in areas presenting alternative states due to different land use histories. Sites that had been clearcut without subsequent use are dominated by the pioneer genus Cecropia, but their understory is characterized by a diverse species assemblage. In contrast, areas clearcut and then used for pasture are dominated by the genus Vismia, forming nearly monogeneric stands. We evaluated whether such patterns were the outcome of differences in community compensatory trends, leading to a dynamic system of sequential replacement of species in Cecropia stands, and to a persistent stage of succession in Vismia stands. Floristic turnover in Cecropia stands showed strong and consistent negative frequency dependence. In contrast, Vismia stands exhibited little or no frequency dependence, likely due to local competitive interactions or priority effects. In these stands, species of the genera Vismia and Bellucia remained dominant throughout the monitoring period, whereas species initially of low abundance and frequency remained so. Differences in recruitment were the major driver of these alternative states. As species colonization proceeds, we expect dominance in the Vismia stands to diminish, albeit slowly. Our approach proved to be a useful tool for comparing species turnover in systems presenting alternative states.     

Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

The cerebrovascular dysfunction induced by slow pressor doses of angiotensin II precedes the development of hypertension

Hypertension alters cerebrovascular regulation and increases the brain's susceptibility to stroke and dementia. We investigated the temporal relationships between the arterial pressure (AP) elevation induced by "slow pressor" angiotensin II (ANG II) infusion, which recapitulates key features of human hypertension, and the resulting cerebrovascular dysfunction. Minipumps delivering saline or ANG II for 14 days were implanted subcutaneously in C57BL/6 mice (n = 5/group). Cerebral blood flow was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. With ANG II (600 ng · kg(-1) · min(-1)), AP started to rise after 9 days (P < 0.05 vs. saline), remained elevated at 11-17 days, and returned to baseline at 21 days (P > 0.05). ANG II attenuated the cerebral blood flow increase induced by neural activity (whisker stimulation) or endothelium-dependent vasodilators, an effect observed before the AP elevation (7 days), as well as after the hypertension subsided (21 days). Nonpressor doses of ANG II (200 ng · kg(-1) · min(-1)) induced cerebrovascular dysfunction and oxidative stress without elevating AP (P > 0.05 vs. saline), whereas phenylephrine elevated AP without inducing cerebrovascular effects. ANG II (600 ng · kg(-1) · min(-1)) augmented neocortical reactive oxygen species (ROS) with a time course similar to that of the cerebrovascular dysfunction. Neocortical application of the ROS scavenger manganic(I-II)meso-tetrakis(4-benzoic acid)porphyrin or the NADPH oxidase peptide inhibitor gp91ds-tat attenuated ROS and cerebrovascular dysfunction. We conclude that the alterations in neurovascular regulation induced by slow pressor ANG II develop before hypertension and persist beyond AP normalization but are not permanent. The findings unveil a striking susceptibility of cerebrovascular function to the deleterious effects of ANG II and raise the possibility that cerebrovascular dysregulation precedes the elevation in AP also in patients with ANG II-dependent hypertension.     

Multivariate analysis of bacterial volatile compound profiles for discrimination between selected species and strains in vitro

Volatile compounds (VCs) are produced by all microorganisms as part of their normal metabolism. The aim of this study was to determine whether bacterial VC profiles could be used to discriminate between selected bacterial species and strains in vitro.Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) was used to quantify the concentration of 23 microbial VCs within the head-space of various bacterial monocultures, during both the logarithmic and stationary growth phases. In comparison with existing techniques, SIFT-MS enables quantitative, high throughput, real-time head-space analysis to be performed, without need for sample preparation. The results show that most VCs were produced by > 1 bacterial species or strain, and some were produced by all strains tested. Multivariate analysis using similarity matrices, cluster analysis and multidimensional scaling (MDS) was used to determine whether there was a characteristic VC profile at either the species or strain level. Significant discrimination of all bacterial species and strains was achieved by analysing the VC profiles, and the relative similarity of VC profiles could be differentiated in 2 or 3 dimensional space. This study has shown that there are significant differences in the volatile profiles obtained from various bacterial monocultures grown in vitro, and that the analysis techniques herein employed have the potential to differentiate samples at the strain level.     

Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.     

Evaluation of Econem™, a Formulated Pasteuria sp. Bionematicide, for Management of Belonolaimus longicaudatus on Golf Course Turf

In 2010, a turfgrass bionematicide containing in vitro produced Pasteuria sp. for management of Belonolaimus longicaudatus was launched under the tradename Econem^™. Greenhouse pot studies and field trials on golf course fairways and tee boxes evaluated Econem at varied rates and application frequencies. Trials on putting greens compared efficacy of three applications of Econem at 98 kg/ha to untreated controls and 1,3-dichloropropene at 53 kg a.i/ha. Further putting green trials evaluated the ability of three applications of Econem at 98 kg/ha to prevent resurgence of population densities of B. longicaudatus following treatment with 1,3-dichloropropene at 53 kg a.i./ha. None of the Econem treatments in pot studies were effective at reducing B. longicaudatus numbers (P ≤ 0.05). Econem was associated with reduction in population densities of B. longicaudatus (P ≤ 0.1) on only a single sampling date in one of the eight field trials and did not improve turf health in any of the trials (P > 0.1). These results did not indicate that Econem is an effective treatment for management of B. longicaudatus on golf course turf.