Quantifying agonist activity at G protein-coupled receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (τ). The estimate of τK(obs) of one agonist, divided by that of another, is a relative measure of K(b) (RA(i)). For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τ(sys)). In this case, the K(b) value of an agonist is equivalent to τK(obs)/τ(sys). Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.     

Diet restriction and life history trade-offs in short- and long-lived species of Daphnia

The life-extending effects of diet restriction are well documented. One evolutionary model that accounts for this widespread conservation is the resource allocation model, where the selected individuals are those that can delay reproduction during periods of resource limitation. In this study, we use closely related species of a model organism, Daphnia, with widely divergent lifespans to address the relationship between diet restriction and longevity and assess whether the relationships are owing to trade-offs between reproductive and somatic investment. Specifically, we conducted a common garden experiment and constructed reaction norms for lifespan, fecundity, and body size as a function of food concentration. Our study provides evidence that the short-lived species in our study, D. pulex, shows the classically observed relationship of enhanced lifespan in response to reduced diet intake, but does not divert resources to somatic maintenance at the expense of reproduction during chronic diet restriction. In contrast, we find no evidence that the long-lived species in our study, D. pulicaria, gains any life-extending effects through diet restriction. Combined, our results provide evidence that the resource allocation model is not sufficient to explain the evolution of diet-mediated lifespan plasticity.     

有害疣孢霉Hypomyces perniciosus遗传转化体系的建立及其突变体库的构建

有害疣孢霉Hypomyces perniciosus是引起双孢蘑菇Agaricus bisporus湿泡病的病原真菌,目前其致病分子机理尚不清楚,而高效稳定的遗传转化体系和突变体库构建是挖掘和研究病原菌致病基因的基础和有效手段。因此,本实验以高致病力的有害疣孢霉菌株WH001为研究对象,采用冻融法将双元载体pBHt1转入农杆菌AGL-1中,建立并优化根癌农杆菌介导的遗传转化体系,并利用其构建T-DNA插入突变体库。结果表明有害疣孢霉菌株WH001的潮霉素（Hygromycin,Hyg）耐受浓度为250ng/L,当农杆菌侵染液浓度OD₆₀₀=1,侵染时间为30min,乙酰丁香酮（Acetosyringone,AS）浓度为1.5mg/mL,共培养时间为3d时,转化体系效率最高。然后利用该优化体系构建有害疣孢霉的突变体库,通过PCR检测和形态学鉴定获得若干表型发生改变、稳定遗传的T-DNA插入突变体,与原菌种WH001相比,突变体在菌丝形态、生长速率、色素分泌和致病力等方面发生改变。本研究为进一步挖掘有害疣孢霉未知基因功能、解析生物学性状、探讨致病分子机制奠定基础。     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Episomal persistence of recombinant adenoviral vector genomes during the cell cycle in vivo

Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% reduction in transgene expression levels and a 50 and 71% loss of vector genomes for the HD vector and the first-generation adenoviral vector. In sharp contrast, in nonviral, episomal plasmid DNA-injected mice, transgene expression levels and DNA copy numbers decreased by 95 and 99%, respectively. These findings suggest that cell division alone was not the primary reason for the slow decrease in transgene expression levels and that recombinant adenoviral vectors have a more robust mechanism for maintaining persistence during cell cycling. Several potential mechanisms are proposed.     

A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Recent sympatric speciation involving habitat-associated nuptial colour polymorphism in a crater lake cichlid

Even though the idea that modes of speciation other than allopatric speciation are possible in nature is now widespread, compelling examples of ecological speciation in sympatry remain rare. We studied an undescribed radiation of haplochromine cichlids in a young crater lake in western Uganda, and in the small river that is nearby but has currently no known surface connection to the lake. We describe two different modes of speciation that occurred in this cichlid lineage within the past 1,500–10,000 years. Not constrained by gene flow, allopatric divergence between river and lake cichlids affects many different morphological traits as well as nuptial colouration—muted in the river, but intensified and polymorphic in lake cichlids—and neutral genetic differentiation. More surprisingly, we demonstrate a case for sympatric speciation within the small lake that is associated with dramatic differences in male breeding colouration (yellow with bright red-chest versus bright blue) and subtle differences in microhabitat, feeding regime and morphology. Reproductive isolation by assortative mating is suggested by significant differentiation between yellow and blue males in neutral markers of gene flow despite complete sympatry. We hypothesize speciation is mediated by divergent selection on sexual signalling between microhabitats.

     

Strong coupling effects during <Emphasis Type="Italic">X</Emphasis>-pulse CPMG experiments recorded on heteronuclear <Emphasis Type="Italic">ABX</Emphasis> spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as ¹³C _i –¹³C _j –¹H, where ¹³C _i and ¹³C _j are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of ¹H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2′ position, deuterated at all other sugar positions and ¹³C labeled at all sugar carbons is presented to illustrate the problems that strong ¹³C–¹³C coupling introduces and a simple solution is proposed.     

Visual stimuli that elicit appetitive behaviors in three morphologically distinct species of praying mantis

We assessed the differences in appetitive responses to visual stimuli by three species of praying mantis (Insecta: Mantodea), Tenodera aridifolia sinensis, Mantis religiosa, and Cilnia humeralis. Tethered, adult females watched computer generated stimuli (erratically moving disks or linearly moving rectangles) that varied along predetermined parameters. Three responses were scored: tracking, approaching, and striking. Threshold stimulus size (diameter) for tracking and striking at disks ranged from 3.5 deg (C. humeralis) to 7.8 deg (M. religiosa), and from 3.3 deg (C. humeralis) to 11.7 deg (M. religiosa), respectively. Unlike the other species which struck at disks as large as 44 deg, T. a. sinensis displayed a preference for 14 deg disks. Disks moving at 143 deg/s were preferred by all species. M. religiosa exhibited the most approaching behavior, and with T. a. sinensis distinguished between rectangular stimuli moving parallel versus perpendicular to their long axes. C. humeralis did not make this distinction. Stimulus sizes that elicited the target behaviors were not related to mantis size. However, differences in compound eye morphology may be related to species differences: C. humeralis’ eyes are farthest apart, and it has an apparently narrower binocular visual field which may affect retinal inputs to movement-sensitive visual interneurons.