Microbial ecology and long-term persistence of crude oil in a taiga spruce forest

Thesis (Ph.D.) University of Alaska Fairbanks, 1997The microbial ecology of a 1976 experimental crude oil spill in an Alaskan taiga black spruce forest was investigated in this study. Substantial oil residue remained in the soil, and several microbial parameters showed evidence of long-term oiling effects. Overall, the data suggest that the surviving community in the oiled plot has shifted toward using oil C for growth. Numbers of hydrocarbon degrading microbes, and specific hydrocarbon mineralization potentials, were significantly elevated in the oiled (OIL) plot compared to an adjacent oil-free, reference (REF) plot. Glutamate mineralization potentials and soil C mineralization, on the other hand, were not different between treatments, suggesting that OIL plot heterotrophs were well-acclimated to the oil. Despite little difference between OTL and REF soils in total C mineralized in vitro, net N mineralized was lower and net nitrification was absent in OIL soils. Analysis of the residual oil indicated minimal amounts of N were added with the spilled oil. Biomasses of total fungi and bacteria, and numbers of protozoa, showed no consistent effects due to oiling, but metabolically active fungal and bacterial biomasses were uniformally lower in OTL samples. Community-level multiple substrate metabolism (Biolog) was assessed using a new technique for extracting kinetic data from the microplates. This analysis suggested that the microbial population diversity in the OIL soils was lower than in REF soils. Further, these data indicated that the surviving populations in the OIL plot may be considered metabolic generalists. Some evidence of crude oil biodegradation was seen in the chemistry data, but enrichment of the oil residue in higher molecular weight components, duration of contact with soil organic material, and slow rates of C mineralization indicate the crude oil will persist at this site for decades. Contamination of Alaskan taiga soil at this site has yielded observable long-term microbial community effects with larger-scale consequences for ecosystem function

Migrating relational data to an OODB: strategies and lessions from a molecular biology experience

Journal ArticleThe growing maturity of OODB technology is causing many enterprises to consider migrating relational databases to OODBs. While data remapping is relatively straightforward, greater challenges lie in economically and non-invasively adapting legacy application software. We report on a genetics laboratory database migration experiment, which was facilitated by both organization of the relational data in object-like form and a C++ framework designed to insulate application code from relational artifacts. To our surprise, the framework failed to encapsulate three subtle aspects of the relational implementation, thereby "contaminating" application code. We describe the underlying issues, and offer cautionary guidance to future migrators

Homomeric and native α7 acetylcholine receptors exhibit remarkably similar but non-identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex

AbstractSucrose gradient analysis of chick acetylcholine receptor (AChR) α7 subunits expressed in oocytes indicates that they form pharmacologically active homomers of the same size as native α7 AChRs, a size compatible with a complex of five α7 subunits. By immunoisolating the [35S]methionine-labeled α7 subunits we also demonstrate that they do not appear to assemble with endogenous Xenopus AChR subunits. Pharmacological characterization of detergent-solubilized brain α7 AChRs and α7 homomers reveals that they have similar but nonidentical properties. The pharmacological difference is most accentuated for cytisine (~50-fold). Thus, at least in E18 chicken brain, most or all of the native α7 AChRs do not appear to be homomeric

Affinity labelling of neuronal acetylcholine receptors localizes acetylcholine-binding sites to their β-subunits

AbstractNeuronal nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats consist of two types of subunits, α and β, of which a shares some antigenic determinants with α-subunits from AChRs of electric organ and muscle [(1986) Biochemistry 25, 2082-2093; (1986) J. Neurosci. G, 3061-3069; (1986) Proc. Natl. Acad. Sci. USA, in press]. Here we demonstrate that after reduction with dithiothreitol (DTT) the AChRs can be specifically labelled with the acetylcholine-binding site directed reagent 4-(N-maleimido)benzyltri [3H]methylammonium iodide. Labelling of the β-subunits of neuronal nicotinic AChRs indicates that the acetylcholine-binding site, and amino acids which may be homologous to Cys 192–193 of the α-subunits of AChRs from electric organ and muscle, are located on the β-subunit of neuronal AChRs. These results suggest that although neuronal nicotinic AChRs have some structural homologies to AChRs from muscle and electric organs, the AChRs from these sources are quite distant relatives in an extended gene family

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model - recommendations for methods and experimental designs.

Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies

Differential α4(+)/(-)β2 Agonist-Binding Site Contributions To α4β2 Nicotinic Acetylcholine Receptor Function Within And Between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)2(β2)3 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist- binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2∗-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect

Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd “protected” fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501–506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436–462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively

Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis.

Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na(+) -dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family choline-transporter like protein (CTL)1-5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na(+) -independent and CTL1-5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis

Myasthenia gravis-like syndrome induced by expression of interferon gamma in the neuromuscular junction.

Abnormal humoral responses toward motor end plate constituents in muscle induce myasthenia gravis (MG). To study the etiology of this disease, and whether it could be induced by host defense molecules, we examined the consequences of interferon (IFN) gamma production within the neuromuscular junction of transgenic mice. The transgenic mice exhibited gradually increasing muscular weakness, flaccid paralysis, and functional disruption of the neuromuscular junction that was reversed after administration of an inhibitor of acetylcholinesterase, features which are strikingly similar to human MG. Furthermore, histological examination revealed infiltration of mononuclear cells and autoantibody deposition at motor end plates. Immunoprecipitation analysis indicated that a previously unidentified 87-kD target antigen was recognized by sera from transgenic mice and also by sera from the majority of human MG patients studied. These results suggest that expression of IFN-gamma at motor end plates provokes an autoimmune humoral response, similar to human MG, thus linking the expression of this factor with development of this disease