Agonist-mediated regulation of presynaptic receptor function during development of rat septal neurons in culture

Presynaptic receptors modulating the release of acetylcholine (ACh) were studied in fetal septal neurons cultured in a growth medium to which various drugs were added from day 3 in vitro (DIV 3) to DIV 14. The influence of these drugs on the function of the presynaptic muscarinic (M-) autoreceptor was determined at DIV 14 by measuring the inhibitory effect of the M-agonist oxotremorine on the electrically-evoked release of [(3)H]ACh from cultures pre-incubated with [(3)H]choline. The presence of the M-agonists oxotremorine (100 micromol/L) or carbachol (100 micromol/L) from DIV 3 to DIV 14, or from DIV 13 to DIV 14, abolished M-autoreceptor function at DIV 14, whereas the presence of the M-antagonist atropine (10 micromol/L from DIV 3 to DIV 14) during growth left M-autoreceptor function unaltered. Inhibition of ACh esterase by donepezil (1 micromol/L from DIV 3 to DIV 14) weakly decreased M-autoreceptor function at DIV 14; inhibition of neuronal firing by 0.1 tetrodotoxin (0.1 micromol/L from DIV 3 to DIV 14) did not tend to affect M-autoreceptor function at DIV 14. Co-cultivation of fetal septal and raphe neurons for 2 weeks yielded cell cultures containing both vesicular ACh transporter- and tryptophan hydroxylase-immunopositive cells. From these cultures, the release of both [(3)H]ACh and [(3)H]5-HT could be induced by electrical field stimulation. In co-cultured neurons versus septal-only ones the inhibitory effect of oxotremorine on the evoked release of [(3)H]ACh appeared almost normal, whereas that of the selective 5-HT(1B) agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) was completely abolished. The effects of CP-93,129 were also absent on DIV 14 in septal mono-cultures grown in the presence of CP-93,129 (10 micromol/L) from DIV 3 to DIV 14. It is therefore concluded that the regulation of presynaptic receptor function strongly depends on the concentrations of endogenous transmitters in the neuronal environment.     

Urinary Biomarkers at Early ADPKD Disease Stage

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by a decline in renal function at late disease stage when the majority of functional renal parenchyma is replaced by cystic tissue. Thus, kidney function, assessed by estimated glomerular filtration rate (eGFR) does not well represent disease burden in early disease. Here, we investigated various urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course.

Methods

ADPKD patients between 18 and 40 years with an eGFR greater or equal to 70 ml per min per 1.73m² were eligible for this cross-sectional study. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1), and Uromodulin (UMOD) were investigated by Enzyme-Linked Immunosorbent Assay. Clara Cell Protein 16 (CC16) was investigated by Latex Immuno Assay. Cryoscopy was performed to assess urine osmolality and Urinary Albumin-to-Creatinine Ratio (UACR) was calculated. The association and the predictive properties of the markers on eGFR and height adjusted total kidney volume (htTKV) was evaluated using multiple regression analysis, incorporating different control variables for adjustment. Internal bootstrapping validated the obtained results.

Results

In 139 ADPKD patients (age 31 ±7 years, mean eGFR of 93 ± 19 ml per min per 1.73 m²) the total kidney volume was negatively correlated with eGFR and UMOD and positive associated with age, UACR, KIM-1 and urine osmolality after adjustment for possible confounders. Urine osmolality and htTKV were also associated with eGFR, whereas no association of CC16, NGAL and UMOD with eGFR or htTKV was found.

Conclusion

UACR and urinary KIM-1 are independently associated with kidney size but not with renal function in our study population. Urine osmolality was associated with eGFR and kidney volume following adjustment for multiple confounders. Despite statistical significance, the clinical value of our results is not yet conceivable. Further studies are needed to evaluate the property of the aforementioned biomarkers to assess disease state at early ADPKD stage.     

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.     

A framework for modelling soil structure dynamics induced by biological activity

Soil degradation is a worsening global phenomenon driven by socio‐economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil–crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil–plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil–crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.     

Effect of elevating serum lipids on luteinizing hormone response to gonadotrophin releasing hormone challenge in energy-deficient anestrous heifers

A study was conducted to evaluate the effect of feeding a bypass fat on luteinizing hormone (LH) response to gonadotrophin releasing hormone (GnRH) in noncyclic Holstein heifers. Twelve cyclic Holstein heifers were fed a complete diet at 40% net energy for maintenance (NE(m)) until cessation of ovarian activity. Based on weights and condition scores, heifers were assigned to either a control or treatment diet containing 0.45 kg bypass fat and fed at an energy level of 85% NE(m). Diet adjustments were made following weekly weighings. GnRH challenges were conducted at four periods: prior to initial energy deprivation, at termination of 40% NE(m) feeding, and twice more at 21-d intervals after 85% NE(m) feeding began. Blood was sampled via a jugular catheter every 15 min for 5 h, and GnRH was injected after the fourth sample. None of the heifers exhibited estrous activity after the initial energy deprivation. Heifers on the bypass fat diet continued to lose weight during the treatment period, while the control heifers gained a slight amount of weight. Baseline and peak concentrations of LH were not significantly affected by time or diet. Time to GnRH-induced LH peak was longer (53 vs 130 min, P < 0.01) after 40% NE(m) and remained greater at all times thereafter. Serum lipid levels increased 82.5% among heifers being fed the bypass fat. Energy restriction had no effect on the magnitude of LH response to GnRH but did delay response time.     

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.     

Boosting Escherichia coli’s heterologous production rate of ectoines by exploiting the non-halophilic gene cluster from Acidiphilium cryptum

Extremophiles - The compatible solutes ectoine and hydroxyectoine are synthesized by many microorganisms as potent osmostress and desiccation protectants. Besides their successful implementation...     

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.