Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans

A microsomal cytochrome P-450-NADPH dependent enzyme which hydroxylates stereo- and regiospecifically carbon atom 14 of (S)- -N- methyltetrahydroprotoberberines has been discovered in a number of plant cell cultures originating from species containing protopine alkaloids; the monooxygenase was solubilized, partially purified (100-fold) and characterized

Androgen Receptor Modulates Multimodal Displays in the Bornean Rock Frog (Staurois parvus)

Multimodal communication is common in the animal kingdom. It occurs when animals display by stimulating two or more receiver sensory systems, and often arises when selection favors multiple ways to send messages to conspecifics. Mechanisms of multimodal display behavior are poorly understood, particularly with respect to how animals coordinate the production of different signals. One important question is whether all components in a multimodal display share an underlying physiological basis, or whether different components are regulated independently. We investigated the influence of androgen receptors (ARs) on the production of both visual and vocal signal components in the multimodal display repertoire of the Bornean rock frog (Staurois parvus). To assess the role of AR in signal production, we treated reproductively active adult males with the antiandrogen flutamide (FLUT) and measured the performance of each component signal in the multimodal display. Our results show that blocking AR inhibited the production of multiple visual signals, including a conspicuous visual signal known as the foot flag, which is produced by rotating the hind limb above the body. However, FLUT treatment caused no measurable change in vocal signaling behavior, or in the frequency or fine temporal properties of males calls. Our study, therefore, suggests that activation of AR is not a physiological prerequisite to the coordination of multiple signals, in that it either does not regulate all signaling behaviors in a male s display repertoire or it does so only in a context-dependent manner

DO LONGITUDINAL MEASURES OF CLINICAL VARIATION CORRELATE WITH ADHERENCE IN CYSTIC FIBROSIS

Introduction: An electronic clinical record (ECR) coding for all variables of CF and capturing “real time” data from 2007 was used as a basis for this study. From this data, longitudinal patterns of clinical variation have emerged that suggest a relationship between variation in lung function and adherence. Our aim was to determine a) the accuracy of self-report adherence and its relationship with clinical variation, and b) whether objective measures, including clinical variation can predict adherence. Methods: Patients aged 16 years and over attending an adult regional CF centre were invited to complete an adherence questionnaire (CFQ-R) and consent to pharmacy script data collection (previous 6 months). Coefficient of variation for FEV1 (CoV FEV1) was calculated from all contacts within the previous year. Age, gender, microbiology, disease severity (banding status), medication, lung function and weight were noted at baseline. Self-reported adherence was calculated against prescribed medication (using ECR) and patients classified into one of 3 categories: low (80%) according to % calculated adherence. Ordinal regression was used to determine the contribution of age, gender, microbiology status, disease severity (Banding status), medication, genotype and CoV FEV1 to self-reported adherence. Results: Patients (n=250) [age 29.7 (±9.2) y, 58.6% (M), baseline BMI 22.5 kg/m2 (±3.8), FEV1 61.3% (±25.1), FVC 79.4% (±23.1)] completed the study. Pharmacy collection data were available for 106 (42%) patients. Pharmacy script collection was strongly correlated to self-reported adherence (Table). CoV FEV1 was inversely related to self-reported adherence (Table 1). Regression analysis revealed that CoV FEV1 [OR = 0.95; CI: 0.92-0.98, p=0.006], number of types of medication [OR = 1.18; CI: 1.11- 1.26, p<0.001], and age [OR = 1.03; CI: 1.01 to 1.06, p=0.026] together explained 19% of the variance in the model. Conclusion: Self-report adherence consistently exceeds medication collection by an average of 14%. Coefficient variation of FEV1 may be an indirect measure of adherence and contributed to the final adherence model. Accurate longitudinal measures captured in real time can aid in examining adherence and warrant further investigation. Supported by a grant from Gilead Sciences

Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in Gα i Subunits † , ‡

Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4− reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release

Multimodal Communication in a Noisy Environment: A Case Study of the Bornean Rock Frog Staurois parvus

High background noise is an impediment to signal detection and perception. We report the use of multiple solutions to improve signal perception in the acoustic and visual modality by the Bornean rock frog, Staurois parvus. We discovered that vocal communication was not impaired by continuous abiotic background noise characterised by fast-flowing water. Males modified amplitude, pitch, repetition rate and duration of notes within their advertisement call. The difference in sound pressure between advertisement calls and background noise at the call dominant frequency of 5578 Hz was 8 dB, a difference sufficient for receiver detection. In addition, males used several visual signals to communicate with conspecifics with foot flagging and foot flashing being the most common and conspicuous visual displays, followed by arm waving, upright posture, crouching, and an open-mouth display. We used acoustic playback experiments to test the efficacy-based alerting signal hypothesis of multimodal communication. In support of the alerting hypothesis, we found that acoustic signals and foot flagging are functionally linked with advertisement calling preceding foot flagging. We conclude that S. parvus has solved the problem of continuous broadband low-frequency noise by both modifying its advertisement call in multiple ways and by using numerous visual signals. This is the first example of a frog using multiple acoustic and visual solutions to communicate in an environment characterised by continuous noise

Formation and annihilation of nanocavities during keV ion irradiation of Ge

Nanocavities in Ge(111) created by 5 keV Xe ion irradiation are characterized by ex situ transmission electron microscopy and Rutherford backscattering spectrometry. Nanocavities nucleate near the surface and then undergo thermal migration. Nanocavities with average diameter of 10 nm and areal density of 5.1 x 10-3 nm-2 are observed at 773 K, while nanocavities with average diameter of 2.9 nm and areal density of 3.1 x 10-3 nm-2 are observed at 673 K. The estimated Xe gas pressure inside the nanocavities is 0.035 GPa at 773 K, much smaller than the estimated equilibrium pressure 0.38 GPa. This result suggests that the nanocavities grow beyond equilibrium size at 773 K. The nanocavities are annihilated at the surface to form surface pits by the interaction of displacement cascades of keV Xe ions with the nanocavities. These pits are characterized by in situ scanning tunneling microscopy. Pits are created on Ge(111) and Ge(001) at temperatures ~ 523-578 K by keV Xe ions even when less than a bilayer (monolayer) of surface material is removed.Comment: 26 pages, 7 figures, to be published in Physical Review

Evolution of a Signaling Nexus Constrained by Protein Interfaces and Conformational States

Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies

Unifying view of mechanical and functional hotspots across class A GPCRs

G protein-coupled receptors (GPCRs) are the largest superfamily of signaling proteins. Their activation process is accompanied by conformational changes that have not yet been fully uncovered. Here, we carry out a novel comparative analysis of internal structural fluctuations across a variety of receptors from class A GPCRs, which currently has the richest structural coverage. We infer the local mechanical couplings underpinning the receptors' functional dynamics and finally identify those amino acids whose virtual deletion causes a significant softening of the mechanical network. The relevance of these amino acids is demonstrated by their overlap with those known to be crucial for GPCR function, based on static structural criteria. The differences with the latter set allow us to identify those sites whose functional role is more clearly detected by considering dynamical and mechanical properties. Of these sites with a genuine mechanical/dynamical character, the top ranking is amino acid 7x52, a previously unexplored, and experimentally verifiable key site for GPCR conformational response to ligand binding. \ua9 2017 Ponzoni et al

Divergent receiver responses to components of multimodal signals in two foot-flagging frog species.

Multimodal communication of acoustic and visual signals serves a vital role in the mating system of anuran amphibians. To understand signal evolution and function in multimodal signal design it is critical to test receiver responses to unimodal signal components versus multimodal composite signals. We investigated two anuran species displaying a conspicuous foot-flagging behavior in addition to or in combination with advertisement calls while announcing their signaling sites to conspecifics. To investigate the conspicuousness of the foot-flagging signals, we measured and compared spectral reflectance of foot webbings of Micrixalus saxicola and Staurois parvus using a spectrophotometer. We performed behavioral field experiments using a model frog including an extendable leg combined with acoustic playbacks to test receiver responses to acoustic, visual and combined audio-visual stimuli. Our results indicated that the foot webbings of S. parvus achieved a 13 times higher contrast against their visual background than feet of M. saxicola. The main response to all experimental stimuli in S. parvus was foot flagging, whereas M. saxicola responded primarily with calls but never foot flagged. Together these across-species differences suggest that in S. parvus foot-flagging behavior is applied as a salient and frequently used communicative signal during agonistic behavior, whereas we propose it constitutes an evolutionary nascent state in ritualization of the current fighting behavior in M. saxicola