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Biomedical subjects

A G Spencer

Publications and source records attributed to A G Spencer.

17 recordsLinked to original sources
PubMed2001

A RHO GTPase-mediated pathway is required during P cell migration in Caenorhabditis elegans.

The Rho family of guanine triphosphate hydrolases controls various cellular processes, including cell migration. We describe here the demonstration of a role for a RhoA GTPase homologue during cell migration in Caenorhabditis elegans. We show that eliminating or reducing rho-1 gene function by using a dominant-negative transgene or dsRNA interference results in a severe defect in migration of hypodermal P cells to a ventral position. Biochemical and genetic data also suggest that unc-73, which encodes a Trio-like guanine nucleotide exchange factor, may act as an activator of rho-1 in the migration process. Mutations in let-502 ROCK, a homologue of a RhoA effector in mammals, also cause defects in P cell migration, suggesting that it may be one of several effectors acting downstream of rho-1 during P cell migration. Finally, we provide evidence to support the idea that other small Rac subfamily small GTPases act redundantly and in parallel to RHO-1 in this specific cell migration event.

Animals↗
PubMed2001

The status and development of insecticide resistance in Danish populations of the housefly Musca domestica L.

Samples of housefly (Musca domestica) field populations were collected from Danish livestock farms in 1997. The tolerance of the first-generation offspring was determined for a number of insecticides. Dose-response values were obtained by topical application for the pyrethroids bioresmethrin and pyrethrum, both synergised with piperonyl butoxide, and the organophosphate dimethoate. The organophosphates azamethiphos and propetamphos and the carbamate methomyl were tested in discriminating dose feeding bioassays. Resistance was low to moderate in most of the populations for most of the compounds tested, but this study also revealed the existence of high resistance to pyrethroid, organophosphate and carbamate insecticides in some populations. The resistance factors at LD50 for bioresmethrin/piperonyl butoxide ranged between 2 and 98, and for pyrethrum/piperonyl butoxide between 2 and 29. Our results indicate that pyrethroid resistance in Denmark is increasing, since four of the 21 farms showed more than 100-fold resistance at LD95, a level of resistance only observed once before. Resistance factors at LD50 for dimethoate ranged from 9 to 100, and showed two distinct trends: populations with either decreasing or increasing resistance. Resistance to azamethiphos was found to be widespread and high. Although two strains with high methomyl and propetamphos resistance were observed, methomyl and propetamphos resistance is moderate and appears not to be increasing.

Animals↗
PubMed2000

Selection and reversion of azamethipos-resistance in a field population of the housefly Musca domestica (Diptera: Muscidae), and the underlying biochemical mechanisms.

The organophosphorus insecticide, azamethiphos, is widely used throughout the world to control the housefly, Musca domestica (L.). Since its commercial introduction to Denmark in 1983 for this purpose, we have monitored the toxicity of azamethiphos to housefly populations at livestock farms throughout the country and carried out regular field studies. The findings of our field studies, which have revealed a strong potential for resistance development, have been born out by regular surveys showing that resistance has increased in recent years. Through the analysis of a field derived laboratory strain, we have implicated oxidative and hydrolytic mechanisms together with altered acetylcholinesterase in this resistance. Our field and laboratory studies have also indicated that resistance is relatively unstable, and can revert in the absence of selection. The implications of our findings for the continued efficacy of azamethiphos are discussed.

Acetylcholinesterase↗
PubMed1999

The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis.

Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.

Amino Acid Sequence↗
PubMed1998

Subcellular localization of prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs like aspirin and ibuprofen. These enzymes catalyze the committed step in the formation of prostanoids from arachidonic acid. Although PGHS-1 and -2 are similar biochemically, a number of studies suggest that PGHS-1 and PGHS-2 function independently to form prostanoids that subserve different cellular functions. We have hypothesized that these isozymes may reside, at least in part, in different subcellular compartments and that their compartmentation may affect their access to arachidonic acid and serve to separate the functions of the enzymes. To obtain high resolution data on the subcellular locations of PGHS-1 and -2, we employed immunoelectron microscopy with multiple antibodies specific to each isozyme. Both PGHS-1 and -2 were found on the lumenal surfaces of the endoplasmic reticulum (ER) and nuclear envelope of human monocytes, murine NIH 3T3 cells, and human umbilical vein endothelial cells. Within the nuclear envelope, PGHS-1 and -2 were present on both the inner and outer nuclear membranes and in similar proportions. Western blotting data showed a similar distribution of PGHS-1 and -2 in subcellular fractions, and product analysis using isozyme-specific inhibitors suggested that both enzymes generate the same products in NIH 3T3 cells. Thus, we are unable to attribute the independent functioning of PGHS-1 and PGHS-2 to differences in their subcellular locations. Instead, the independent operation of these isozymes may be attributable to subtle kinetic differences (e.g. negative allosteric regulation of PGHS-1 at low concentrations of arachidonate (500-1000 nM)). A further conclusion of importance from a cell biological perspective is that membrane proteins such as PGHS-1 and -2, which are located on the lumenal surface of the ER, are able to diffuse freely among the ER and the inner and outer membranes of the nuclear envelope.

3T3 Cells↗
PubMedDate not supplied

The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks.

We evaluated the role of beetles infesting broiler chicken rearing facilities as potential reservoirs for Salmonella enterica infections between successive broiler flocks. In addition, their role as potential reservoirs for thermophilic Campylobacter spp. was also investigated. Fourteen broiler houses located at 11 different farms were included in the study. The houses were nonrandomly selected on the basis of their salmonella status; nine were persistently contaminated with salmonella whereas five were salmonella negative. For each broiler house, two consecutive broiler flocks (i.e., 28 broiler flocks in all) as well as beetles collected during both rotations of production and in the empty period (after cleaning and disinfection) between these flocks were monitored for the presence of salmonella. Examinations for the presence of campylobacter in the same sample materials were also performed. Beetles sampled during production were positive for salmonella or campylobacter or both. Furthermore, in one house, the occurrence of Salmonella indiana in two consecutive broiler flocks coincided with the presence of S. indiana-contaminated beetles in the empty period between the flocks. The genotype of the identified S. indiana was in all cases identical when analyzed by pulsed-field gel electrophoresis. However, our results also suggest that salmonella from beetles may not always be transmitted to the chickens and that beetles living in contaminated houses can remain free of infection. All cases of campylobacter-positive beetle samples were detected in connection with a positive chicken flock; in no case was campylobacter isolated from beetles taken from the empty period between rotations. Four beetle species were identified during this study. Alphitobius diaperinus was found in all houses and was relatively abundant in most. Typhaea stercorea and Ahasverus advena were found in eight and nine houses, respectively, and were abundant in most of these. Carcinops pumilio was found in small numbers in eight houses. No other insect species was identified. These investigations have shown that beetles in broiler houses infrequently are positive for salmonella. However, transmission of S. indiana between two consecutive broiler flocks can coincide with the presence of salmonella-contaminated beetles in the empty period, indicating that the beetles were the reservoir of S. indiana between the two flocks. Concerning campylobacter, the results suggest that beetles do not play a significant role as a reservoir of campylobacter from one rotation to the next.

Animal Husbandry↗