Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands

Background  

G-protein-coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However, purification of GPCRs for biochemical study is difficult and current methods of studying receptor-ligand interactions involve in vitro systems. Caenorhabditis elegans is a soil-dwelling, bacteria-feeding nematode that uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds, making this an ideal system for studying in vivo GPCR-ligand interactions. We sought to test this by functionally expressing two medically important mammalian GPCRs, somatostatin receptor 2 (Sstr2) and chemokine receptor 5 (CCR5) in the gustatory neurons of C. elegans.     

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors.

The role of muscarinic receptors in the down-regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10(-6) M) and atropine (10(-8) to 10(-6) M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10(-6) M) caused a slight depression in the amplitude of responses to ACh pressure-applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA-immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10(-6) M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10(-6) M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10(-6) M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse.     

The anatomy of Lepetella sierrai (Vetigastropoda,Lepetelloidea): implications for reproduction,feeding, and symbiosis in lepetellid limpets

The Lepetelloidea, a clade of small limpet‐shaped gastropods, can be used as a case study in continental margin and deep‐sea diversification. Lineages in this clade have been found associated with a combination of different substrates, including hydrothermal vents, seeps, wood, whale carcasses, polychaete tubes, chondrichthyan egg cases, seagrass rhizomes, algal holdfasts, crab carapaces, and sponges. Members of one lepetelloidean family, Lepetellidae, live on or inside empty tubes of members of the polychaete genus Hyalinoecia. The detailed morphology of a Mediterranean species, Lepetella sierrai Dantart & Luque 1944, was reconstructed in three dimensions from serial semi‐thin sections and compared with that of eleven other members of Lepetellidae. The hermaphroditic lepetellid limpets possessed a ciliated seminal groove, distinct testis and ovary with a common distal gonoduct, and a seminal receptacle containing mature sperm. A unique alimentary tract, with huge esophageal pouches, no true stomach, an extensive multilobed midgut, and short intestine, was present. Additionally, a bacteriocyte system throughout the entire mantle rim was revealed via light and transmission electron microscopy. This is the first recognized evidence for intracellular microbial symbiosis in lepetelloidean limpets. Semi‐thin sections showed evidence of a parasite, potentially a chitonophilid copepod, penetrating the body wall of the limpet. Hypotheses about reproductive biology, feeding, and symbiosis are presented based on anatomical features and knowledge of the habitat described herein.     

Improved analyses of human mtDNA sequences support a recent African origin for Homo sapiens

New quantitative methods are applied to the 135 human mitochondrial sequences from the Vigilant et al. data set. General problems in analyzing large numbers of short sequences are discussed, and an improved strategy is suggested. A key feature is to focus not on individual trees but on the general "landscape" of trees. Over 1,000 searches were made from random starting trees with only one tree (a local optimum) being retained each time, thereby ensuring optima were found independently. A new tree comparison metric was developed that is unaffected by rearrangements of trees around many very short internal edges. Use of this metric showed that downweighting hypervariable sites revealed more evolutionary structure than studies that weighted all sites equally. Our results are consistent with convergence toward a global optimum. Crucial features are that the best optima show very strong regional differentiation, a common group of 49 African sequences is found in all the best optima, and the best optima contain the 16 !Kung sequences in a separate group of San people. The other 86 sequences form a heterogeneous mixture of Africans, Europeans, Australopapuans, and Asians. Thus all major human lineages occur in Africa, but only a subset occurs in the rest of the world. The existence of these African-only groups strongly contradicts multiregional theories for the origin of Homo sapiens that require widespread migration and interbreeding over the entire range of H. erectus. Only when the multiregional model is rejected is it appropriate to consider the root, based on a single locus, to be the center of origin of a population (otherwise different loci could give alternative geographic positions for the root). For this data, several methods locate the root within the group of 49 African sequences and are thus consistent with the recent African origin of H. sapiens. We demonstrate that the time of the last common ancestor cannot be the time of major expansion in human numbers, and our results are thus also consistent with recent models that differentiate between the last common ancestor, expansion out of Africa, and the major expansion in human populations. Such a two-phase model is consistent with a wide range of molecular and archeological evidence.      

Hemolymph lipids and flight activity in Helicoverpa zea (Boddie) (lepidoptera: Noctuidae)

In this study the flight activity of female and male Helicoverpa zea (Boddie) moths was observed and compared to hemolymph lipid concentrations. The major male and female H. zea flight activity occurred between simulated dusk (1700) and dawn (0300). Male flight activity was up to 7 times greater than females through 6 days after eclosion except for the 1st day (0.8 times). Females had a unimodal pattern of flight activity, peaking between dusk and 2 h later. Males had a bimodal pattern; one between dusk and 2 h later, and another 3 h after dusk, continuing for h. Prior to dusk, total neutral hemolymph lipids (neutral) of H. zea day 4 moths was 64 μg/μl for males and 48 μg/μl for females. Typical lipid composition in day 4 males prior to flight was 1,2-diacylglycerides (DG) (50% w/w), triacylglycerides (TG) (35%), cholesterol esters (2%), and less than 1% monoacylglycerides and cholesterol. The remainder consisted of free fatty acids (<0.5 μg/μl), and various uncharacterized phospholipids and lipophilic compounds. Hemolymph DG concentration patterns were similar between day 4 males and females, were highest in both sexes prior to, during, and after flight (approximately 32 μg/μl), and then decreased steadily throughout the flight period to approximately 16 μg/ml as flight ceased. Hemolymph TG were lower than DG, but followed the same pattern except at 2100 and 2300. In day 4 males between 2100 and 2300, TG increased to 33 μg/μl which was when DG was lowest (15 μg/μl) and their flight activity was highest. Hemolymph DG decreased (26 to 20 μg/μl) in day 4 females between 2100 and 2300 as TG remained fairly constant (18 μg/μl).     

Utilisation by homeless people of acute hospital services in London.

OBJECTIVES--To estimate the numbers and distribution of homeless people in London; to quantify the utilisation of acute inpatient services by homeless people in two health authorities; and to predict the total numbers of admissions in homeless people in district health authorities across London. DESIGN--Data were collected from various sources on the distribution of homeless people across London boroughs. All unplanned acute inpatient admissions during November 1990 to relevant hospitals were identified. SETTING--Bloomsbury and Paddington and North Kensington, two former inner London district health authorities. SUBJECTS--Homeless people in London residing in bed and breakfast and private sector leased accommodation, residing in hostels, and of no fixed abode. MAIN OUTCOME MEASURES--Number and cost of acute unplanned admissions in homeless people in two health authorities in November 1990; predicted number of such admissions each year in district health authorities in London. RESULTS--There were at least 60,000 homeless people in London in March 1990. The majority were housed in temporary accommodation (55,412). There were at least 3295 hostel dwellers and 651 people sleeping rough. Homeless people accounted for 105 (8%) of the 1256 acute unbooked admissions in residents of Bloomsbury and Paddington and North Kensington health authorities in November 1990. Considerable variations in the pattern of acute unplanned admissions in homeless people were observed in the two districts with respect to housing status and specialty of admission. The total number of acute unplanned admissions in homeless people across London each year was estimated at 7598, ranging from 38 in Bexley to 1515 in Parkside. CONCLUSIONS--The results have fundamental implications for resource allocation across London. Allocation must take better account of the heterogeneity, uneven distribution, and extra health needs of homeless people.