Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Assessing the Suitability of Various Physiological Traits to Screen Wheat Genotypes for Salt Tolerance

Success of improving the salt tolerance of genotypes requires effective and reliable screening traits in breeding programs. The objective was to assess the suitability of various physiological traits to screen wheat genotypes for salt tolerance. Thirteen wheat genotypes from Egypt, Germany, Australia and India were grown in soil with two salinity levels （control and 150 mmol/L NaCI） in a greenhouse. The physiological traits （ion contents in leaves and stems, i.e. Na^＋, Cl^-, K^＋, Ca^2＋）, the ratios of K^＋/Na^＋ and Ca^＋/Na^＋ in the leaves and stems, net photosynthesis rate, stomatal conductance, transpiration rate, chlorophyll content （SPAD value）, and leaf water relations, were measured at different growth stages. The physiological traits except for Na^＋ and Cl^- in stems and the leaf transpiration rate at 150 mmol/L NaCI showed a significant genotypic variation, indicating that the traits that have a significant genotypic variation may be possibly used as screening criteria. According to the analysis of linear regression of the scores of the physiological traits against those of grain yield, however, the physiological traits of Ca^2＋ and Ca2^＋/Na^＋ at 45 d and final harvest with the greatest genotypic variation were ranked at the top. From a practical and economic point of view, SPAD value should be considered to be used as screening criteria and/or there is a need to develop a quick and practical approach to determine Ca^2＋ in plant tissues.     

Bacteria and phosphorus regeneration in lakes. An experimental study

The aerobic decomposition of the green alga Chlamydomonas reinhardii by a mixed population of lake bacteria was studied in batch and chemostat cultures. Bacterial chemostats were supplied with continuously heatkilled algae. The dead algae rapidly released most of their phosphorus as SRP. In the batch experiments bacteria acted as consumers of the released algal phosphorus. This phosphorus uptake was dependent on the C:P ratio of the algae. During the death phase of the bacteria most of the bacterial phosphorus itself was released. The continuous supply of energy in form of dead algae in the chemostat experiments prevented the death phase of the bacteria and thus any net regeneration of phosphorus. The influence of the C:P stoichiometry of algae and bacteria on the regeneration of algal phosphorus is discussed.     

Altered mechanisms of sympathetic activation during rhythmic forearm exercise in heart failure

In congestive heart failure (CHF), themechanisms of exercise-induced sympathoexcitation are poorly defined.We compared the responses of sympathetic nerve activity directed tomuscle (MSNA) and to skin (SSNA, peroneal microneurography) duringrhythmic handgrip (RHG) at 25% of maximal voluntary contraction andduring posthandgrip circulatory arrest (PHG-CA) in CHF patients with those of an age-matched control group. During RHG, the CHF patients fatigued prematurely. At end exercise, the increase in MSNA was similarin both groups (CHF patients, n = 12;controls, n = 10). However, duringPHG-CA, in the controls MSNA returned to baseline, whereas it remainedelevated in CHF patients (P < 0.05).Similarly, at end exercise, the increase in SSNA was comparable in bothgroups (CHF patients, n = 11;controls, n = 12), whereas SSNAremained elevated during PHG-CA in CHF patients but not in the controls (P < 0.05). In a separate controlgroup (n = 6), even high-intensity static handgrip was not accompanied by sustained elevation of SSNAduring PHG-CA. ³¹P-nuclear magneticresonance spectroscopy during RHG demonstrated significant muscleacidosis and accumulation of inorganic phosphate in CHF patients(n = 7) but not in controls(n = 9). We conclude that in CHFpatients rhythmic forearm exercise leads to premature fatigue andaccumulation of muscle metabolites. The prominent PHG-CA response ofMSNA and SSNA in CHF patients suggests activation of the musclemetaboreflex. Because, in contrast to controls, in CHF patients bothMSNA and SSNA appear to be under muscle metaboreflex control, themechanisms and distribution of sympathetic outflow during exerciseappear to be different from normal.

     

Exposure to moderate air pollution during late pregnancy and cord blood cytokine secretion in healthy neonates

Background/Objectives

Ambient air pollution can alter cytokine concentrations as shown in vitro and following short-term exposure to high air pollution levels in vivo. Exposure to pollution during late pregnancy has been shown to affect fetal lymphocytic immunophenotypes. However, effects of prenatal exposure to moderate levels of air pollutants on cytokine regulation in cord blood of healthy infants are unknown.

Methods

In a birth cohort of 265 healthy term-born neonates, we assessed maternal exposure to particles with an aerodynamic diameter of 10 µm or less (PM₁₀), as well as to indoor air pollution during the last trimester, specifically the last 21, 14, 7, 3 and 1 days of pregnancy. As a proxy for traffic-related air pollution, we determined the distance of mothers'' homes to major roads. We measured cytokine and chemokine levels (MCP-1, IL-6, IL-10, IL-1ß, TNF-α and GM-CSF) in cord blood serum using LUMINEX technology. Their association with pollution levels was assessed using regression analysis, adjusted for possible confounders.

Results

Mean (95%-CI) PM₁₀ exposure for the last 7 days of pregnancy was 18.3 (10.3–38.4 µg/m³). PM₁₀ exposure during the last 3 days of pregnancy was significantly associated with reduced IL-10 and during the last 3 months of pregnancy with increased IL-1ß levels in cord blood after adjustment for relevant confounders. Maternal smoking was associated with reduced IL-6 levels. For the other cytokines no association was found.

Conclusions

Our results suggest that even naturally occurring prenatal exposure to moderate amounts of indoor and outdoor air pollution may lead to changes in cord blood cytokine levels in a population based cohort.     

Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794 bp DNA fragment from Nosema bombycis, 471 bp fragment from B. mori NPV (BmNPV) and 391 bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.     

Antitumor compound testing in glioblastoma organotypic brain cultures

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.     

Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H⁺-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.