Epitope Mapping of Form-Specific and Nonspecific Antibodies to Acetylcholinesterase

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44–82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp⁵⁹, an N-linked glycosylation site not present in mouse AChE. A 20-amino-acid synthetic peptide, RFRRPEPKKPWSQVWNASTY, representing residues 44–63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first α-helix.     

The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.     

Novel limonene and citral based 2,5-disubstituted-1,3,4-oxadiazoles: A natural product coupled approach to semicarbazones for antiepileptic activity

Two novel series of N⁴-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N¹-(2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enylidene)semicarbazide and N⁴-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N¹-(3,7-dimethylocta-3,6-dienylidene)-semicarbazide were synthesized to meet structural prerequisite indispensable for anticonvulsant activity. The anticonvulsant activities of the compounds were investigated using maximal electroshock seizure (MES), subcutaneous pentylenetrtrazole (scPTZ) and subcutaneous strychnine (scSTY) models. The rotorod test was conducted to evaluate neurotoxicity. Some of the selected active compounds were subjected to GABA assay to confirm their mode of action. The outcome of the present investigations proved that the four binding sites pharmacophore model is vital for anticonvulsant activity. The efforts were also made to establish structure–activity relationships among test compounds.     

Characterizing bioavailable phosphorus concentrations in an agricultural stream during hydrologic and streambed disturbances

In freshwater ecosystems, phosphorus (P) is often considered a growth-limiting nutrient. The use of fertilizers on agricultural fields has led to runoff-driven increases in P availability in streams, and the subsequent eutrophication of downstream ecosystems. Isolated storms and periodic streambed dredging are examples of two common disturbances that contribute dissolved and particulate P to agricultural streams, which can be quantified as soluble reactive P (SRP) using the molybdate-blue method on filtered water samples, or total P (TP) measured using digestions on unfiltered water reflecting all forms of P. While SRP is often considered an approximation of bioavailable P (BAP), research has shown that this is not always the case. Current methods used to estimate BAP do not account for the role of biology (e.g., NaOH extractions) or require specialized platforms (e.g., algal bioassays). Here, in addition to routine analysis of SRP and TP, we used a novel yeast-based bioassay with unfiltered sample water to estimate BAP concentrations during two storms (top 80% and?>?95% flow quantiles), and downstream of a reach where management-associated dredging disturbed the streambed. We found that the BAP concentrations were often greater than SRP, suggesting that SRP is not fully representative of P bioavailability. The SRP concentrations were similarly elevated during the two storms, but remained consistently low during streambed disturbance. In contrast, turbidity and TP were elevated during all events. The BAP concentrations were significantly related to turbidity during all disturbance events, but with TP only during storms. The novel yeast assay suggests that BAP export can exceed SRP, particularly when streams are not in equilibrium, such as the rising limb of storms or during active dredging.

     

Biogeochemical transformation of a nutrient subsidy: salmon, streams, and nitrification

Migratory animals can alter ecosystem function via the provision of nutrient subsidies. These subsidies are heterogeneous in space and time, which may create hot spots or hot moments in biogeochemical transformations, in turn altering the ecosystem effect of the subsidy by changing the form of the nutrients. Annual migrations of Pacific salmon (Oncorhynchus spp.) transport nutrients from the marine environment to their natal freshwater ecosystems. Salmon subsidies provide high quality nutrients (e.g., nitrogen, phosphorus, carbon) that may also be large in quantity where salmon migrations are near historic levels. We hypothesized that the nutrient subsidy provided via the excretion of ammonium (NH₄ ⁺) by live salmon would stimulate microbially mediated nitrification rates in stream sediments and increase streamwater nitrate (NO₃ ^?) concentrations. We quantified sediment nitrification in seven streams in Southeast Alaska before and during the salmon run in 2007 and 2008. Nitrification rates increased 3-fold from before to during the salmon run (mean ± SE = 0.07 ± 0.01 to 0.24 ± 0.02 mgN gAFDM^?1 d^?1, respectively). The variation in nitrification was explained by both streamwater and exchangeable NH₄ ⁺ concentrations (R ² = 0.50 and 0.71, respectively), which were low before salmon and increased relative to the size of the salmon run. To experimentally test the effect of salmon subsidies on nitrification rates, we staked senesced salmon carcasses on stream sediments for 3 weeks during the salmon run and then measured nitrification rates directly under the carcasses. Sediment nitrification was 2–5 times higher under the carcasses compared to nearby sediments without the direct carcass influence. Our results confirm that biogeochemical transformations alter the form of salmon-derived nitrogen, representing an overlooked aspect in the dynamics of this subsidy. Therefore, animal-derived nutrient subsidies are not passively retained or exported in recipient ecosystems, but also transformed, thereby influencing the form and incorporation of these nutrient subsidies.     

Determination of serum glucose using co-immobilized glucose oxidase and peroxidase onto arylamine glass beads affixed on a plastic strip

Glucose oxidase (GOD) from Aspergillus niger and horseradish peroxidase (POD) were co-immobilized onto arylamine glass beads affixed on a plastic strip with a conjugation yield of 28.2 mg/g and 43% retention of their initial specific activity. The coimmobilized enzymes showed maximum activity at pH 7.5 when incubated at 37 degrees C for 15 min. A simple, specific and sensitive method for discrete analysis of the serum glucose was developed employing this strip. The minimum detection limit of the method was 5 mg/dl. Within and between assay coefficient of variations for the serum were <5.6% and <10.6% (n = 6) respondely. A good correlation (r = 0.943) was found between the glucose values obtained by the enzyme colorimetric method employing free GOD and POD and the present method. The strip lost 50% of its initial activity after its 150 regular uses for a period of one month, when stored in reaction buffer at 4 degrees C. The method is cost-effective than the enzymic colorimetric method, as the enzyme strip is reusable.     

Quantification of the Nitrogen Cycle in a Prairie Stream

Nitrogen (N) was added for 35 days in the form of ¹⁵NH₄Cl to Kings Creek on Konza Prairie, Kansas. Standing stocks of N in key compartments (that is, nutrients, detritus, organisms) were quantified, and the amount of labeled N entering the compartments was analyzed. These data were used to calculate turnover and flux rates of N cycling through the food web, as well as nutrient transformation rates. Inorganic N pools turned over much more rapidly in the water column of this stream than in pelagic systems where comparable measurements have been made. As with other systems, the mass of ammonium was low but it was the key compartment mediating nutrient flux through the ecosystem, whereas dissolved organic N, the primary component of N flux through the system, is not actively cycled. Nitrification was also a significant flux of N in the stream, with rates in the water column and surface of benthos accounting for approximately 10% of the total ammonium uptake. Primary consumers assimilated 67% of the inorganic N that entered benthic algae and microbes. Predators acquired 23% of the N that consumers obtained. Invertebrate collectors, omnivorous crayfish (Orconectes spp.), and invertebrate shredders dominated the N flux associated with primary consumers. Mass balance calculations indicated that at least 23% of the 309 mg of ¹⁵N added during the 35 days of release was retained within the 210-m stream reach during the release. Overall, the rates of turnover of N in organisms and organic substrata were significantly greater when C:N was low. This ratio may be a surrogate for biological activity with regard to N flux in streams. Received 2 August 1999; accepted 18 July 2000.     

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.