Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC

Faecal near infrared reflectance spectroscopy estimates of diet quality and responses to nitrogen supplements by cattle grazing Bothriochloa pertusa pastures

A grazing experiment in the seasonally dry tropics of north Queensland examined the diet selected and the growth responses of Bos indicus steers to urea supplement over two dry seasons and one wet season, from August 2001 to January 2003. There were two groups of 10 steers (control and urea-supplemented) and each group comprised two age cohorts, A and B, of five steers each with an age difference of ~1 year. In June 2002, cohort A steers were replaced with steers 2 years younger (cohort C). The steers grazed Bothriochloa pertusa pastures on a low fertility Red Chromosol soil. The groups were switched between two adjoining paddocks at fortnightly intervals when they were weighed and faecal samples were collected for faecal near infrared reflectance spectroscopy (F.NIRS) estimates of diet quality and growth rate. Rainfall and diet quality followed the expected seasonal pattern, but the 2001–02 wet season was very short (November–January) with only 65% of the long-term average rainfall. There was no rain during the 2001 dry season (DS-1) from August to mid November, and no effective rain (17 mm) between February and December 2002. Non-grass (i.e. herbaceous dicot plants) made only a small contribution to the diet, averaging 13%. In DS-1 and in the dry season of 2002 (DS-2) diet crude protein (CP) averaged 2.5% and 2.9%, and DMD/CP (ratio of dry matter digestibility to CP) averaged 18.6 and 17.1, respectively. Liveweight (LW) loss in control steers during DS-1 averaged 32 kg. Urea supplement reduced LW loss by 18 kg (P < 0.001), but most of the benefit was lost during the following growing season. During the extended dry period in 2002, average LW losses of control steers were 85 and 47 kg in the older and younger cohorts, respectively, and supplementation with urea reduced weight losses by 53 and 31 kg, respectively (P < 0.001). F.NIRS predicted the cumulative LW of the unsupplemented steers in cohorts A and B with acceptable accuracy, the difference between the observed and predicted final LW being 6 kg for cohort A after 10 months, 12 kg for cohort B after 17 months, and 27 kg for cohort C after 7 months. The results demonstrated that F.NIRS can be effectively used to monitor dietary CP and DMD levels in grazing cattle, to help develop an understanding of cattle responses to urea supplement relative to the quality of the basal forage diet, and to provide useful decision support information for the nutritional management of grazing cattle

Safety in New Zealand weed biocontrol a nationwide pathogen survey for impacts on nontarget plants

The accuracy of host range testing in weed biocontrol programmes using plant pathogens has been questioned prompting a research programme into the field safety record of pathogens used for weed biocontrol in New Zealand Nationwide disease surveys were conducted from 20002009 and focused on species closely related to target weeds and therefore potentially most at risk of attack These surveys identified pathogens associated with any disease symptoms observed on nontarget hosts Disease damage attributable to biocontrol agents was observed on two nontarget plants Pustules of the blackberry rust Phragmidium violaceum were found on the endemic Rubus species R cissoides (bush lawyer tataramoa) at one location This result was predicted from host range safety tests conducted prior to its arrival in New Zealand No nontarget damage was observed in the remaining case studies confirming to date that all biocontrol pathogens are highly host specific to their target weeds</jats:p

Shy Elitism: A New Keyword in Critical Multiculturalism Studies

Since the official promulgation of multiculturalism by Prime Minister Pierre Elliott Trudeau on October 8, 1971, there has been a concerted attempt to build awareness and acceptance of the policy by recognizing the achievements of individuals from immigrant and racialized groups. This chapter draws on extensive archival research to address the work of key figures who advocated for civil liberties, human rights and race relations in Canada within a context of state-sanctioned multiculturalism that uses prestigious annual awards to advance harmonious race relations . It proposes a reading of their strategic interventions in a Canadian public sphere as a form of shy elitism that grants prestigious awards and credentials to 'accessible' work that represses material that may appear too elitist or radical for 'ordinary Canadians'.</p