@article{alarcon_hernandez_garcia_ziarelli_gutierrez-rivera_musule_vazquez-marrufo_gardner_2022, title={Changes in chemical and structural composition of sugarcane bagasse caused by alkaline pretreatments [Ca(OH)(2) and NaOH] modify the amount of endoglucanase and beta-glucosidase produced by Aspergillus niger in solid-state fermentation}, volume={209}, ISSN={["1563-5201"]}, DOI={10.1080/00986445.2021.1881777}, abstractNote={The aim of this study was to evaluate the treatment effects of Ca(OH)2 solutions applied at different concentrations (2.5%, 5%, and 7.5%) on the structural and chemical composition of sugarcane bagasse (SCB). The feasibility of using the SCB as a substrate for endoglucanase and β-glucosidase production by Aspergillus niger through solid-state fermentation was also assessed. Sodium hydroxide (NaOH) 5% was used as a positive control/benchmark. Changes in SCB cell wall compositions was determined using 13C solid-state nuclear magnetic resonance. While cellulosic fiber content and the production of endoglucanase (345.01 ± 139 U.gDM−1) increased using Ca(OH)2 as pretreatment for sugarcane bagasse, hemicellulose, lignin, alkyl C and carboxyl C content decreased. Comparisons between pretreated and non-pretreated SCB revealed that the highest values of β-glucosidase (58.18 ± 9.86 U.gDM−1) occurred in non-pretreated samples. The SCB pretreated with NaOH resulted in the highest delignification values (-49.88%) and the greatest hemicellulose remotion (-53.47%). However, this treatment also increased crystallinity index (CI; + 23.61%) in cellulose and showed the lowest endoglucanase (22.79 ± 11 U.gDM−1) and β-glucosidase (4.71 ± 1.44 U.gDM−1) activities. This study claims that cellulose CI, hemicellulose, and other nutrients (e.g., amino acids) are important variables that should be considered along with delignification, in the substrates selection for cellulase production using filamentous fungi.}, number={5}, journal={CHEMICAL ENGINEERING COMMUNICATIONS}, author={Alarcon, Enrique and Hernandez, Christian and Garcia, Gabriela and Ziarelli, Fabio and Gutierrez-Rivera, Beatriz and Musule, Ricardo and Vazquez-Marrufo, Gerardo and Gardner, Terrence G.}, year={2022}, month={May}, pages={594–606} }
@article{dick_gardner_frene_heitman_sucre_leggett_2022, title={Forest floor manipulation effects on the relationship between aggregate stability and ectomycorrhizal fungi}, volume={505}, ISSN={["1872-7042"]}, DOI={10.1016/j.foreco.2021.119873}, abstractNote={Forest floor and mineral soil manipulations influence the soil biogeochemical properties important for loblolly pine (Pinus taeda L.) tree growth. The impacts of forest floor manipulations on soil aggregate stability and the presence of ectomycorrhizal fungi (EMF), was assessed to elucidate the relationship between EMF abundance and aggregate stability. The study site consists of a 14-year-old loblolly pine plantation managed by Weyerhaeuser Company in the Lower Coastal Plain, approximately 8 miles east of New Bern, North Carolina, USA. The soil samples were collected from the top 7.62 cm of each soil treatment which includes three levels of forest floor retention: removed, control, and doubled and two levels of forest floor mixing with the mineral soil: mixed and unmixed. Ectomycorrhizal fungi abundance was evaluated by ester-linked fatty acid methyl ester analysis and microbial community functionality was assessed by acid-phosphatase activity measurement. Aggregate stability was assessed using the aggregate mean weight diameter approach. Results indicate that the forest floor manipulations had no significant impact on aggregate stability and EMF abundance. However, a positive relationship between EMF abundance and aggregate stability was identified. Removing the forest floor resulted in a soil bulk density increase of 0.18 g cm−3 compared to doubling the forest floor. Our results demonstrate that some mineral soil properties recover relatively quickly from forest floor manipulations. The study informs forest managers interested in how soil responds to forest floor manipulation and the interaction between EMF and aggregate stability.}, journal={FOREST ECOLOGY AND MANAGEMENT}, author={Dick, David L. and Gardner, Terrence G. and Frene, Juan P. and Heitman, Joshua L. and Sucre, Eric B. and Leggett, Zakiya H.}, year={2022}, month={Feb} }
@article{frene_frazier_liu_clark_parker_gardner_2021, title={Early Effect of Pine Biochar on Peach-Tree Planting on Microbial Community Composition and Enzymatic Activity}, volume={11}, ISSN={["2076-3417"]}, url={https://doi.org/10.3390/app11041473}, DOI={10.3390/app11041473}, abstractNote={Biochar offers several benefits as a soil amendment, including increased soil fertility, carbon sequestration, and water-holding capacity in nutrient-poor soils. In this study, soil samples with and without biochar additives were collected for two consecutive years from an experimental field plot to examine its effect on the microbial community structure and functions in sandy soils under peach-trees (Prunus persica). The four treatments evaluated consisted of two different rates of biochar incorporated into the soil (5%, and 10%, v/v), one “dynamic” surface application of biochar, and a 0% biochar control. Fatty acid methyl ester (FAME) analysis was used to assess the microbial community structure, and enzyme activities involved in C, N, P, and S nutrient cycling were used as a means of assessing soil functionality. Total FAME and bacterial indicators increased by 18% and 12%, respectively, in the 10% incorporated and 5% surface applied treatments. Biochar applications increased β-glucosaminidase and arylsulfatase activities, 5–30% and 12–46%, respectively. β-glucosidase and acid phosphatase activities decreased by approximately 18–35% and 5–22% in the 0–15 cm soils. The overall results suggest that biochar’s addition to the sandy soils stimulated microbial activity, contributing to the increased mean weight diameter (MWD), C sequestration, and consequential soil health. The changes in microbial community structure and functions may be useful predictors of modifications in soil organic matter (SOM) dynamics due to the long-term application of pine biochar in these systems.}, number={4}, journal={APPLIED SCIENCES-BASEL}, author={Frene, Juan Pablo and Frazier, Mattie and Liu, Shuang and Clark, Bernadette and Parker, Michael and Gardner, Terrence}, year={2021}, month={Feb} }
@article{vepraskas_amoozegar_gardner_2021, title={Estimation of Saprolite Thickness Needed to Remove E. coli from Wastewater}, volume={11}, ISSN={["2076-3417"]}, url={https://doi.org/10.3390/app11052066}, DOI={10.3390/app11052066}, abstractNote={Saprolite, weathered bedrock, is being used to dispose of domestic sewage through septic system drainfields, but the thickness of saprolite needed to remove biological contaminants is unknown for most saprolites. This study developed and tested a simple method for estimating the thickness of saprolite needed below septic drainlines to filter E. coli from wastewater using estimates of the volume of pores that are smaller than the length of the coliform (≤10 μm). Particle size distribution (texture) and water retention data were obtained for 12 different saprolites from the Piedmont and Mountain regions of North Carolina (N.C.). Saprolite textures ranged from clay loam to coarse sand. The volume of pores with diameters ≤10 μm were determined by water retention measurements for each saprolite. The data were used in an equation to estimate the saprolite thickness needed to filter E. coli. The estimated saprolite thicknesses ranged from 36 cm in the clay loam to 113 cm for the coarse sand. The average thickness across all samples was 58 cm. Saprolite thickness estimates increased as silt percentage decreased and as sand percentage and in situ saturated hydraulic conductivity increased. Silt percentage may be most useful for estimating appropriate saprolite thicknesses in the field.}, number={5}, journal={APPLIED SCIENCES-BASEL}, publisher={MDPI AG}, author={Vepraskas, Michael J. and Amoozegar, Aziz and Gardner, Terrence}, year={2021}, month={Mar} }
@article{alarcon gutierrez_hernandez_gardner_garcia perez_caballero_perroni_silva_gaime perraud_barois_2021, title={Soil bioindicators associated to different management regimes of Cedrela odorata plantations}, volume={27}, ISSN={["2448-7597"]}, url={https://doi.org/10.21829/myb.2021.2711912}, DOI={10.21829/myb.2021.2711912}, abstractNote={A good bioindicator of soil health is one that perceives minor changes due to changes in soil management and leads to changes in the processes of the soil ecosystem. The aim was to evaluate the use of soil bioindicators in the determination of the utility of two different Cedrela odorata forestry management systems. The litter and soil samples were collected from the rhizosphere of a monoculture (with no fertilization nor irrigation), and a co-culture (with fertilization and irrigation) stand with Citrus latifolia. The soil and litter bioindicators measured were the activity of acetylesterase (FDA), acid phosphatase (AcPh), alkaline phosphatase (AlkPh), laccase, and the estimation of the potential net rate of carbon mineralization (PNRCM). The results indicated that soil enzyme activities (FDA and phosphatases) in the litter were sensitive to the different management system treatments. Enzymatic activity, per unit dry weight, was higher in litter of co-culture (FDA = 1.05 nkat g-1 AcPh = 1.33 nkat g-1; AlkPh = 03.0 nkat g-1) than in litter of monoculture (FDA = 0.617nkat g-1; AcPh = 0.40 nkat g-1; AlkPh = 0. 983 nkat g-1). Differences in soil total nitrogen content, organic matter (OM) content, and micronutrients were found. In general, the co-culture stand (with fertilization and irrigation) with C. latifolia showed the best results.}, number={1}, journal={MADERA Y BOSQUES}, publisher={Instituto de Ecologia, A.C.}, author={Alarcon Gutierrez, Enrique and Hernandez, Christian and Gardner, Terrence and Garcia Perez, Jose Antonio and Caballero, Mayra and Perroni, Yareni and Silva, Anne Marie A. and Gaime Perraud, Isabelle and Barois, Isabelle}, year={2021} }
@article{park_liu_gardner_johnson_keeler_ortiz_rabah_ford_2020, title={Biohybrid nanofibers containing manganese oxide-forming fungi for heavy metal removal from water}, volume={15}, ISSN={["1558-9250"]}, DOI={10.1177/1558925019898954}, abstractNote={Manganese-oxidizing fungi support bioremediation through the conversion of manganese ions into manganese oxide deposits that in turn adsorb manganese and other heavy metal ions from the environment. Manganese-oxidizing fungi were immobilized onto nanofiber surfaces to assist remediation of heavy metal–contaminated water. Two fungal isolates, Coniothyrium sp. and Coprinellus sp., from a Superfund site (Lot 86, Farm Unit #1) water treatment system were incubated in the presence of nanofibers. Fungal hyphae had strong association with nanofiber surfaces. Upon fungal attachment to manganese chloride–seeded nanofibers, Coniothyrium sp. catalyzed the conformal deposition of manganese oxide along hyphae and nanofibers, but Coprinellus sp. catalyzed manganese oxide only along its hyphae. Fungi–nanofiber hybrids removed various heavy metals from the water. Heavy metal ions were adsorbed into manganese oxide crystalline structure, possibly by ion exchange with manganese within the manganese oxide. Hybrid materials of fungal hyphae and manganese oxides confined to nanofiber-adsorbed heavy metal ions from water.}, journal={JOURNAL OF ENGINEERED FIBERS AND FABRICS}, author={Park, Yaewon and Liu, Shuang and Gardner, Terrence and Johnson, Drake and Keeler, Aaron and Ortiz, Nathalia and Rabah, Ghada and Ford, Ericka}, year={2020}, month={Jan} }
@article{frene_frazier_rutto_jones_liu_clark_parker_gardner_2020, title={Early response of organic matter dynamics to pine‐biochar in sandy soil under peach trees}, url={https://doi.org/10.1002/agg2.20094}, DOI={10.1002/agg2.20094}, abstractNote={Abstract Biochar may enhance soil health in sandy soils under peach [ Prunus persica (L.) Batsch] tree production. A study was initiated in the Sandhills of North Carolina by applying pine‐biochar at two different rates (5 and 10%, v/v) and types of application (incorporation to 30 cm [‐Inc] and surface [‐Sur]) in 2017. Soil organic matter (SOM) dynamics was assessed via soil organic carbon (SOC), total nitrogen (TN), permanganate‐oxidizable carbon (POXC), aggregate fractions (macro‐, micro‐aggregates, and clay + silt particles), and enzyme activities (CNPS activity) at 0–15 and 15–30 cm in 2018 and 2019. After the first year of pine‐biochar application, POXC decreased in all biochar treatments, SOC was higher in the 10%‐Inc treatment, whereas CNPS activities and macroaggregates were highest in the 5%‐Sur compared with the non‐treated control. These results suggest early changes in SOM dynamics and biogeochemical cycling and have the potential to guide producers with the long‐term application of pine‐biochar to sandy soils.}, journal={Agrosystems, Geosciences & Environment}, author={Frene, Juan Pablo and Frazier, Mattie and Rutto, Edwin and Jones, McGwire and Liu, Shuang and Clark, Bernadette and Parker, Michael and Gardner, Terrence G.}, year={2020}, month={Jan} }
@article{gardner_vepraskas_amoozegar_2020, title={Efficiency of saprolite for removing E. coli from simulated wastewater}, volume={10}, url={http://dx.doi.org/10.2166/wst.2020.514}, DOI={10.2166/wst.2020.514}, abstractNote={Abstract Saprolite, weathered bedrock, is being used to dispose of domestic sewage through septic system drainfields, but its ability to remove coliforms is unknown. This study determined if Escherichia coli could be removed by a sandy loam saprolite material. Triplicate columns containing saprolite were prepared with lengths of 30, 45, and 60 cm. A 215-mL solution containing 1 × 105 CFU/100 mL of non-toxic E. coli was applied to the top of each column for 5 days/week for 13 weeks, and selected outflow samples were analyzed for E. coli. Control columns had only tap water applied to them at the same time. Significantly higher (p ≤ 0.10 compared to controls) E. coli concentrations were only detected in samples collected at the end of week 3 for the 30-cm columns and week 4 for the 45-cm columns. E. coli concentrations were small and ranged from approximately 2 to 3 MPN/100 mL. No E. coli were detected in any outflow from the 60-cm columns. From weeks 5 to 13, E. coli concentrations from all columns were either undetectable or not significantly different from the control. The results showed that 60 cm of sandy loam saprolite was sufficient for the removal of E. coli from simulated wastewater.}, journal={Water Science and Technology}, publisher={IWA Publishing}, author={Gardner, T. and Vepraskas, M. J. and Amoozegar, A.}, year={2020}, month={Oct} }
@article{enzymatic hydrolysis of an organic sulfur compound_2019, url={http://dx.doi.org/10.4236/aer.2019.71001}, DOI={10.4236/aer.2019.71001}, abstractNote={Sulfatases which cleave sulfate esters in biological systems are key enzymes that deserve special attention due to their significant roles in organic sulfur (OS) mineralization and inorganic sulfur () release. In this study, in-vitro experiments were conducted to evaluate S bonded substrate hydrolysis by a commercially available arylsulfatase (EC 3.1.6.1) from Aerobacter aerogenes. The enzyme-substrate interactions were assessed to determine: 1) rate of hydrolysis, 2) catalytic efficiency, 3) thermal stability, and 4) optimal pH of this enzyme. Arylsulfatase exhibited substrate hydrolysis with a high affinity for p-nitrophenyl sulfate (potassium 4-nitrophenyl sulfate (pNPS)). The optimum activity for the enzyme was observed to occur at a pH of 7.1. The optimal temperature was 37°C but ranged from 35°C - 45°C. The apparent Km and Kcat of the enzyme for pNPS hydrolysis at the optimal pH, and temperature were determined to be 1.03 mM and 75.73 μM/min, respectively. This work defines the catalytic and kinetic properties of arylsulfatase (EC 3.1.6.1) and confirms the optimal conditions for sulfatase activity testing. The resulting information is useful in elucidating the contributions that individual enzymes have for specific reactions rather than relying on traditional total enzyme activity measurements.}, year={2019} }
@inproceedings{relationships between nutrient content, microbial communities, and presence of siderophores in north carolina soils._2019, url={https://scisoc.confex.com/scisoc/2019sssa/meetingapp.cgi/Paper/115671}, year={2019}, month={Jan} }
@article{rivers_weber_gardner_liu_armstrong_2018, title={ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis [version 1; referees: awaiting peer review]}, volume={7}, url={https://f1000research.com/articles/7-1418/v1}, DOI={10.12688/f1000research.15704.1}, abstractNote={The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU’s) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.}, number={1418}, journal={F1000Research}, publisher={F1000 Research, Ltd.}, author={Rivers, Adam R. and Weber, Kyle C. and Gardner, Terrence G. and Liu, Shuang and Armstrong, Shalamar D.}, year={2018}, month={Sep}, pages={1418} }
@article{ntoko_gardner_2018, title={Microbial Compositions and Enzymes of a Forest Ecosystem in Alabama: Initial Response to Thinning and Burning Management Selections}, url={https://www.scirp.org/journal/PaperInformation.aspx?PaperID=86043}, journal={Journal of Forestry}, author={Ntoko, F.A. and Gardner}, year={2018}, month={Jul} }
@article{duckworth_rivera_gardner_andrews_santelli_polizzotto_2017, title={Morphology, structure, and metal binding mechanisms of biogenic manganese oxides in a superfund site treatment system}, volume={19}, ISSN={["2050-7895"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85010739340&partnerID=MN8TOARS}, DOI={10.1039/c6em00525j}, abstractNote={Manganese oxides, which may be biogenically produced in both pristine and contaminated environments, have a large affinity for many trace metals.}, number={1}, journal={ENVIRONMENTAL SCIENCE-PROCESSES & IMPACTS}, author={Duckworth, O. W. and Rivera, N. A. and Gardner, T. G. and Andrews, M. Y. and Santelli, C. M. and Polizzotto, M. L.}, year={2017}, month={Jan}, pages={50–58} }
@inbook{duckworth_franzluebbers_gardner_2016, place={SSSA, Madison, WI}, title={Biogeochemical Processes Underpin Ecosystem Services}, DOI={10.2136/2015.soilecosystemsservices.2015.0024.}, booktitle={Soil Ecosystems Services}, author={Duckworth, O.W. and Franzluebbers, A.J. and Gardner, T.G.}, editor={Stromberger, M. and Comerford, N. and Lindbo, D.Editors}, year={2016} }
@article{duckworth_rivera_gardner_andrews_santelli_polizzotto_2016, title={Morphology, Structure, and Metal Binding Mechanisms of Biogenic Manganese Oxides in a Superfund Site Treatment System}, volume={19}, journal={Duckworth, O. W., Rivera, N.A., Gardner, T.G., Andrews, M.Y., Santelli, C.M., and Polizzotto, M.L. 2016. Morphology, Structure, and Metal Binding Mechanisms of Biogenic Manganese Oxides in a Superfund Site Treatment System. Environmental Science: Process & Impacts 19:50-58}, author={Duckworth, O.W. and Rivera, N.A. and Gardner, T.G. and Andrews, M.Y. and Santelli, C.M. and Polizzotto, M.L.}, year={2016}, pages={50–58} }
@article{padilla_calderon_acosta-martinez_van pelt_gardner_baddock_zobeck_noveron_2014, title={Diffuse-reflectance mid-infrared spectroscopy reveals chemical differences in soil organic matter carried in different size wind eroded sediments}, volume={15}, ISSN={["2212-1684"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84908288231&partnerID=MN8TOARS}, DOI={10.1016/j.aeolia.2014.06.003}, abstractNote={Soil organic matter (SOM) is essential for soil water holding capacity, aggregation, and biodiversity. Little information is available regarding the carbon (C) functional groups carried away in wind eroded sediments away from the source soil. Mid-infrared (MidIR) spectroscopy was used on wind tunnel-blown sediments eroded from a loam soil during the fallow period of different cropping systems and tillage managements in Akron, Colorado. The soil was managed as fallow-winter wheat (Triticum aestivum L.) under conventional tillage (F–Wct) or no tillage (F–Wnt) and fallow–wheat–corn under no tillage (F–W–Cnt). Two wind eroded sediments were evaluated: fine dust (<35 μm mean dia.) and saltation-size material (<175 μm mean dia.). Our study showed that there is a partition of C groups within wind eroded sediments of different sizes and that they can reflect the tillage management history of soil. The fine dust had higher levels of aliphatic CH (2930 cm−1), and clays (3690–3620 cm−1). The saltation-sized material showed higher absorbance for quartz from 2000–1800 cm−1 and reduced absorbance from 1250–1050 cm−1. Both wind eroded sediments showed higher absorbance for –OH/NH groups and aliphatic CH from no-till soil. Finer dust sediments, which travel greater distances from the source soil than saltation size material, can carry away higher levels of aliphatic-carbon compounds and clays with potential negative impacts on SOM quantity and quality, and consequently the sustainability of these agroecosystems.}, journal={AEOLIAN RESEARCH}, author={Padilla, Julio E. and Calderon, Francisco J. and Acosta-Martinez, Veronica and Van Pelt, Scott and Gardner, Terrence and Baddock, Matthew and Zobeck, Ted M. and Noveron, Juan C.}, year={2014}, month={Dec}, pages={193–201} }
@article{acosta-martinez_cotton_gardner_moore-kucera_zak_wester_cox_2014, title={Predominant bacterial and fungal assemblages in agricultural soils during a record drought/heat wave and linkages to enzyme activities of biogeochemical cycling}, volume={84}, ISSN={["1873-0272"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84904319460&partnerID=MN8TOARS}, DOI={10.1016/j.apsoil.2014.06.005}, abstractNote={Identification of microbial assemblages predominant under natural extreme climatic events will aid in our understanding of the resilience and resistance of microbial communities to climate change. From November 2010 to August 2011, the Southern High Plains (SHP) of Texas, USA, received only 39.6 mm of precipitation (vs. the historical average of 373 mm) and experienced the three hottest months (June–August 2011) since record keeping began in 1911. The objective of this study was to characterize soil bacterial (16 S rRNA gene) and fungal (internal transcribed spacer 1–4, ITS1-ITS4) species distribution and diversity via pyrosequencing during the peak of the drought/heat wave in July 2011 and when the Drought Index and temperatures were lower in March 2012. Samples were collected from two different soil types (loam and sandy loam) under two different dryland cropping histories (monoculture vs. rotation). Fungal Diversity Indexes were significantly higher after the drought/heat wave while Bacterial Indexes were similar. Bacterial phyla distribution in July 2011 was characterized by lower relative abundance of Acidobacteriaand Verrucomicrobia, and greater relative abundance of Proteobacteria, Chloroflexi, Actinobacteria and Nitrospirae than March 2012 samples. Further grouping of pyrosequencing data revealed approximately equal relative proportions of Gram positive (G+) and Gram negative (G−) bacteria in July 2011, while G− bacteria predominated in March 2012. Fungal class Dothideomycetes was approximately two times greater in July 2011 than in March 2012, while the class Sordariomycetes and a group of unidentified OTUs from Ascomycota increased from July 2011 to March 2012. Microbial community composition was less influenced by management history than by the difference in climatic conditions between the sampling times. Correspondence analysis identified assemblages of fungal and bacterial taxa associated with greater enzyme activities (EAs) of C, N, or P cycling found during the drought/heat wave. Microbial assemblages associated with arylsulfatase activity (key to S cycling), which increased after the drought/heat wave, were identified (Streptomyces parvisporogenes, Terrimonas ferruginea and Syntrophobacter sp.) regardless of the soil and management history. The distinct microbial composition found in July 2011 may represent assemblages essential to maintaining ecosystem function during extreme drought and intense heat waves in semiarid agroecosystems.}, journal={APPLIED SOIL ECOLOGY}, author={Acosta-Martinez, V. and Cotton, J. and Gardner, T. and Moore-Kucera, J. and Zak, J. and Wester, D. and Cox, S.}, year={2014}, month={Dec}, pages={69–82} }
@article{acosta-martinez_moore-kucera_cotton_gardner_wester_2014, title={Soil enzyme activities during the 2011 Texas record drought/heat wave and implications to biogeochemical cycling and organic matter dynamics}, volume={75}, ISSN={["1873-0272"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84888420330&partnerID=MN8TOARS}, DOI={10.1016/j.apsoil.2013.10.008}, abstractNote={Extreme droughts and heat waves due to climate change may have permanent consequences on soil quality and functioning in agroecosystems. During November 2010 to August 2011, the Southern High Plains (SHP) region of Texas, U.S., a large cotton producing area, received only 39.6 mm of precipitation (vs. the historical avg. of 373 mm) and experienced the hottest summer since record keeping began in 1911. Several enzyme activities (EAs) important in biogeochemical cycling were evaluated in two soils (a loam and a sandy loam at 0–10 cm) with a management history of monoculture (continuous cotton) or rotation (cotton and sorghum or millet). Samplings occurred under the most extreme drought and heat conditions (July 2011), after precipitation resulted in a reduction in a drought severity index (March 2012), and 12 months after the initial sampling (July 2012; loam only). Eight out of ten EAs, were significantly higher in July 2011 compared to March 2012 for some combinations of soil type and management history. Among these eight EAs, enzymes key to C (β-glucosidase, β-glucosaminidase) and P cycling (phosphodiesterase, acid and alkaline phosphatases) were significantly higher (19–79%) in July 2011 than in March 2012 for both management histories regardless of the soil type (P > 0.05). When comparing all sampling times, the activities of alkaline phosphatase, aspartase and urease (rotation only) showed this trend: July 2011 > March 2012 > July 2012. Activities of phosphodiesterase, acid phosphatase, α-galactosidase, β-glucosidase and β-glucosaminidase were higher in July 2011 than July 2012 in at least one of the two management histories. Total C was reduced significantly from July 2011 to March 2012 in the rotation for both soils. Only the activities of arylsulfatase (avg. 36%) and asparaginase showed an increase from July 2011 to March 2012 for both soil types, which may indicate they have a different origin/location than the other enzymes. EAs continued to be a fingerprint of the soil management history (i.e., higher EAs in the rotation than in monoculture) during the drought/heat wave. This study provided some of the first evidence of the adverse effects of a natural, extreme drought and heat wave on soil quality in agroecosystems as indicated by EAs involved in biogeochemical cycling.}, journal={APPLIED SOIL ECOLOGY}, author={Acosta-Martinez, V. and Moore-Kucera, J. and Cotton, J. and Gardner, T. and Wester, D.}, year={2014}, month={Mar}, pages={43–51} }
@article{a workshop for developing learning modules for science classes based on biogeochemical research_2013, journal={Natural Sciences Education}, year={2013}, month={May} }
@article{gardner_acosta-martinez_calderón_zobeck_baddock_van pelt_senwo_dowd_cox_2012, title={Pyrosequencing reveals bacteria carried in different wind-eroded sediments}, volume={41}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84862266252&partnerID=MN8TOARS}, DOI={10.2134/jeq2011.0347}, abstractNote={Little is known about the microbial communities carried in wind-eroded sediments from various soil types and land management systems. The novel technique of pyrosequencing promises to expand our understanding of the microbial diversity of soils and eroded sediments because it can sequence 10 to 100 times more DNA fragments than previous techniques, providing enhanced exploration into what microbes are being lost from soil due to wind erosion. Our study evaluated the bacterial diversity of two types of wind-eroded sediments collected from three different organic-rich soils in Michigan using a portable field wind tunnel. The wind-eroded sediments evaluated were a coarse sized fraction with 66% of particles >106 μm (coarse eroded sediment) and a finer eroded sediment with 72% of particles <106 μm. Our findings suggested that (i) bacteria carried in the coarser sediment and fine dust were effective fingerprints of the source soil, although their distribution may vary depending on the soil characteristics because certain bacteria may be more protected in soil surfaces than others; (ii) coarser wind-eroded sediment showed higher bacterial diversity than fine dust in two of the three soils evaluated; and (iii) certain bacteria were more predominant in fine dust (, , and ) than coarse sediment ( and ), revealing different locations and niches of bacteria in soil, which, depending on wind erosion processes, can have important implications on the soil sustainability and functioning. Infrared spectroscopy showed that wind erosion preferentially removes particular kinds of C from the soil that are lost via fine dust. Our study shows that eroded sediments remove the active labile organic soil particulates containing key microorganisms involved in soil biogeochemical processes, which can have a negative impact on the quality and functioning of the source soil.}, number={3}, journal={Journal of Environmental Quality}, author={Gardner, T. and Acosta-Martinez, V. and Calderón, F.J. and Zobeck, T.M. and Baddock, M. and Van Pelt, R.S. and Senwo, Z. and Dowd, S. and Cox, S.}, year={2012}, pages={744–753} }
@article{gardner_acosta-martinez_senwo_dowd_2011, title={Soil rhizosphere microbial communities and enzyme activities under organic farming in Alabama}, volume={3}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84859391710&partnerID=MN8TOARS}, DOI={10.3390/d3030308}, abstractNote={Evaluation of the soil rhizosphere has been limited by the lack of robust assessments that can explore the vast complex structure and diversity of soil microbial communities. Our objective was to combine fatty acid methyl ester (FAME) and pyrosequencing techniques to evaluate soil microbial community structure and diversity. In addition, we evaluated biogeochemical functionality of the microbial communities via enzymatic activities of nutrient cycling. Samples were taken from a silt loam at 0–10 and 10–20 cm in an organic farm under lettuce (Lactuca sativa), potato (Solanum tuberosum), onion (Allium cepa L), broccoli (Brassica oleracea var. botrytis) and Tall fescue pasture grass (Festuca arundinacea). Several FAMEs (a15:0, i15:0, i15:1, i16:0, a17:0, i17:0, 10Me17:0, cy17:0, 16:1ω5c and 18:1ω9c) varied among the crop rhizospheres. FAME profiles of the soil microbial community under pasture showed a higher fungal:bacterial ratio compared to the soil under lettuce, potato, onion, and broccoli. Soil under potato showed higher sum of fungal FAME indicators compared to broccoli, onion and lettuce. Microbial biomass C and enzyme activities associated with pasture and potato were higher than the other rhizospheres. The lowest soil microbial biomass C and enzyme activities were found under onion. Pyrosequencing revealed significant differences regarding the maximum operational taxonomic units (OTU) at 3% dissimilarity level (roughly corresponding to the bacterial species level) at 0–10 cm (581.7–770.0) compared to 10–20 cm (563.3–727.7) soil depths. The lowest OTUs detected at 0–10 cm were under broccoli (581.7); whereas the lowest OTUs found at 10–20 cm were under potato (563.3). The predominant phyla (85%) in this soil at both depths were Bacteroidetes (i.e., Flavobacteria, Sphingobacteria), and Proteobacteria. Flavobacteriaceae and Xanthomonadaceae were predominant under broccoli. Rhizobiaceae, Hyphomicrobiaceae, and Acidobacteriaceae were more abundant under pasture compared to the cultivated soils under broccoli, potato, onion and lettuce. This study found significant differences in microbial community structure and diversity, and enzyme activities of nutrient cycling in this organic farming system under different rhizospheres, which can have implications in soil health and metabolic functioning, and the yield and nutritional value of each crop.}, number={3}, journal={Diversity}, author={Gardner, T. and Acosta-Martinez, V. and Senwo, Z. and Dowd, S.E.}, year={2011}, pages={308–328} }
@article{bhatnagar_gülland_bascand_palmer_gardner_kearse_bäckström_2003, title={Mutational analysis of conserved amino acids in the T cell receptor α-chain transmembrane region: A critical role of leucine 112 and phenylalanine 127 for assembly and surface expression}, volume={39}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0345701216&partnerID=MN8TOARS}, DOI={10.1016/S0161-5890(03)00027-0}, abstractNote={Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.}, number={15}, journal={Molecular Immunology}, author={Bhatnagar, A. and Gülland, S. and Bascand, M. and Palmer, E. and Gardner, T.G. and Kearse, K.P. and Bäckström, B.T.}, year={2003}, pages={953–963} }
@article{gardner_kearse_2000, title={Purification of immature CD4+CD8+ thymocytes by panning with anti-CD8 antibody.}, volume={134}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033643752&partnerID=MN8TOARS}, journal={Methods in molecular biology (Clifton, N.J.)}, author={Gardner, T.G. and Kearse, K.P.}, year={2000}, pages={47–53} }
@article{gardner_franklin_robinson_pederson_howe_kearse_2000, title={T cell receptor assembly and expression in the absence of calnexin}, volume={378}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034214002&partnerID=MN8TOARS}, DOI={10.1006/abbi.2000.1804}, abstractNote={Most subunits of the αβδϵγϵζζ T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRα,β molecules and invariant CD3γ,δ,ϵ chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRα subunits, the role of calnexin in the survival and assembly of remaining TCR components is unknown. Here we evaluated the expression of TCR proteins in CEM T cells and the calnexin-deficient CEM variant CEM.NKR. We found that CEM and CEM.NKR cells constitutively synthesized all TCR subunits except for TCRα and that CD3-γ,δ,ϵ components and CD3-β complexes were effectively assembled together in both cell types. The stability and folding of core CD3ϵ chains were similar in CEM and CEM.NKR cells. Interestingly, TCRα synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NKR cells and TCRα proteins synthesized in CEM.NKR cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NKR cells in the absence of calnexin synthesis. These results demonstrate that TCR complexes can be expressed in the absence of calnexin and suggest that the role of calnexin in the quality control of TCR assembly is primarily restricted to the stabilization of newly synthesized TCRα proteins.}, number={1}, journal={Archives of Biochemistry and Biophysics}, author={Gardner, T.G. and Franklin, R.A. and Robinson, P.J. and Pederson, N.E. and Howe, C. and Kearse, K.P.}, year={2000}, pages={182–189} }
@article{gardner_kearse_1999, title={Modification of the T cell antigen receptor (TCR) complex by UDP- glucose:glycoprotein glucosyltransferase: TCR folding is finalized convergent with formation of αβδεγε complexes}, volume={274}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033553467&partnerID=MN8TOARS}, DOI={10.1074/jbc.274.20.14094}, abstractNote={Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing α, β, γ, δ, ε, and ζ molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3αβ chains and formation of CD3-associated, disulfide-linked αβ heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of αβδεγε complexes. Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing α, β, γ, δ, ε, and ζ molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3αβ chains and formation of CD3-associated, disulfide-linked αβ heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of αβδεγε complexes. The antigen receptor expressed on most T lymphocytes is the multisubunit αβ T cell receptor complex (TCR), 1The abbreviations used are: TCR, T cell antigen receptor; ER, endoplasmic reticulum; GT, UDP-glucose:glycoprotein glucosyltransferase; mAb, monoclonal antibody; dmj, deoxymannojirimycin; chx, cycloheximide; PAGE, polyacrylamide gel electrophoresis; EH, endoglycosidase H important for recognition of major histocompatibility complex molecules containing bound peptides (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). The αβTCR is composed of six distinct proteins: clonotypic TCRα and -β molecules and invariant CD3γ, -δ, -ε, and -ζ chains (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). TCR assembly is initiated in the endoplasmic reticulum (ER) and occurs via the ordered pairing of: (i) CD3γ, -δ, and -ε chains into partial complexes of δε and γε components; (ii) association of clonotypic proteins with CD3 chains to form αδε and βγε intermediate complexes; (iii) joining of αδε and βγε molecules to create incomplete αβδεγε complexes, within which disulfide linkage of α and β chains occurs; and finally, (iv) addition of ζζ homodimers to form complete αβδεγεζζ complexes (2Exley M Terhorst C. Wileman T. Semin. Immunol. 1991; 3: 283-297PubMed Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). In most T cell types, intracellular transport and expression of TCR proteins is tightly regulated by their assembly status. Unassembled and partially assembled TCR proteins are retained within the ER and disposed of by poorly understood mechanisms involving retrograde transport to the cytosol and degradation by proteasomes (4Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 5Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 6Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Incomplete (αβδεγε) and complete (αβδεγεζζ) TCR complexes egress from the ER to the Golgi; however, incomplete TCR complexes are sorted to lysosomes where they are degraded. Only complete TCR complexes efficiently traffic to the cell surface (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). Four TCR subunits are post-translationally modified by addition of oligosaccharides TCRα (3 N-glycans), TCRβ (4N-glycans), CD3δ (3 N-glycans), and CD3γ (1N-glycan) (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). N-Glycan chains on newly translated proteins have the structure Glc3Man9GlcNAc2 and are sequentially processed by glucosidase I and II ER enzymes to form monoglucosylated Glc1Man9GlcNAc2 species, important for interaction with the endogenous lectins calnexin and calreticulin that function in the quality control system of protein folding (7Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-634Crossref PubMed Scopus (3779) Google Scholar, 8Ware F.E. Vassilakos A. Peterson P.A. Jackson M.A. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 10Spiro R.G. Zhu Q. Bhoyroo V. Soling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar); the final, innermost Glc residue is removed by glucosidase II (gII) before or after chaperone disassembly. Fully trimmed (Glc0) proteins that persist in a malfolded state are modified by UDP-glucose:glycoprotein glucosyltransferase (GT), which transfers a single Glc residue, (re)creating monoglucosylated (Glc1) species that can (re)enter the calnexin, calreticulin assembly pathway (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). GT is proposed to be a major sensor of protein folding in the ER (11Gannan S. Cazzulo J.J. Parodi A.J. Biochemistry. 1991; 30: 3098-3104Crossref PubMed Scopus (52) Google Scholar, 12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 13Fernandez F.S. Trombetta S.E. Hellman U. Parodi A.J. J. Biol. Chem. 1994; 269: 30701-30706Abstract Full Text PDF PubMed Google Scholar) and will only add back Glc residues removed by gII if a glycoprotein has not yet acquired its proper tertiary structure (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). The deglucosylation/reglucosylation cycle continues until correct conformation is achieved (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). GT modification of incompletely folded proteins involves interaction with both polypeptide and glycan determinants, including recognition of hydrophobic amino acids and interestingly, the innermost GlcNAc residue of the glycan chain (the site of attachment of oligosaccharide to protein) (15Sousa M.C. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (242) Google Scholar). Both recognition elements must be covalently linked to effectively catalyze Glc transfer (15Sousa M.C. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (242) Google Scholar) and accessible to GT modification, which for certain glycoproteins may be concealed by molecular chaperone association in vivo, particularly under conditions of extreme ER stress (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). The size of the glycan chain,e.g. the oligomannose core, is also important for the efficiency of reglucosylation; Man8–9GlcNAc2glycans are reglucosylated much more effectively than shorter Man5–7GlcNAc2 glycans (12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). As recently demonstrated in mutant BW cell types synthesizing truncated Glc3Man5GlcNAc2 N-glycans, TCRα molecules having shortened oligosaccharides were reglucosylated much less efficiently than TCRα molecules having normal size glycans, which was correlated with TCRα instability (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To evaluate the folding status of TCR glycoproteins as a function of their assembly into multisubunit complexes in the ER, we studied the GT modification of TCR proteins in 2B4 T hybridoma cells. These studies show that all TCR subunits bearing N-glycan chains were modified by GT and that TCR proteins were differentially reglucosylated during their assembly into multisubunit complexes. Furthermore, these data demonstrate that reglucosylation of most TCR subunits was extinguished following CD3αβ assembly and formation of CD3-associated disulfide-linked αβ heterodimers, indicating that TCR folding is finalized convergent with formation of αβδεγε complexes. 2B4 T hybridoma cells and the TCRβ-deficient 2B4 variant 21.2.2 were maintained by weekly passage in RPMI 1640 medium containing 5% fetal calf serum at 37 °C in 5% CO2 (17Hedrick S.M. Matis L.A. Hecht T.T. Samelson L.E. Longo D.L. Heber-Katz E. Schwartz R.H. Cell. 1982; 30: 141-152Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 18Samelson L.E. Germain R.M. Schwartz R.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6972-6976Crossref PubMed Scopus (230) Google Scholar, 19Sussman J.J. Saito T. Shevach E.M. Germain R.N. Ashwell J.D. J. Immunol. 1988; 140: 2520-2526PubMed Google Scholar). The following monoclonal antibodies (mAb) were used in this study: H57-597, specific for TCRβ proteins (20Kubo R.T. Born W. Kappler J.W. Marrack P. Pigeon M. J. Immunol. 1989; 142: 2736-2743PubMed Google Scholar); A2B4 specific for 2B4 TCRα proteins (18Samelson L.E. Germain R.M. Schwartz R.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6972-6976Crossref PubMed Scopus (230) Google Scholar); 145-2C11 specific for CD3γε, δε molecules (21Leo O. Foo M. Sachs D.J. Samelson L.E. Bluestone J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1374-1378Crossref PubMed Scopus (1704) Google Scholar); HMT3.2, which recognizes both murine CD3γ and CD3δ proteins (22Dietrich J. Neisig A. Hou X. Wegener A.M.K. Gajhede M. Geisler C. J. Cell Biol. 1996; 132: 299-310Crossref PubMed Scopus (66) Google Scholar); the following antiserum was used: R9, specific for CD3δ molecules (23Samelson L.E. Weissman A.M. Robey F.A. Berkower I. Klausner R.D. J. Immunol. 1986; 137: 3254-3258PubMed Google Scholar). Deoxymannojirimycin (dmj) was purchased from Roche Molecular Biochemicals and was used at a final concentration of 75 μg/ml. Metabolic pulse-labeling with [3H]galactose was performed as described previously (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Briefly, cells were incubated in glucose-free RPMI 1640 medium (Life Technologies, Inc.) containing 10% dialyzed fetal calf serum, 5 mm sodium pyruvate (Life Technologies, Inc.), and 1 mm cycloheximide (chx) for 3 min at 37 °C in 5% CO2; cells were centrifuged and resuspended in similar medium containing 0.5 mCi/ml ([6-3H]galactose) (ICN, Irvine, CA) and labeled for 15–45 min at 37 °C in 5% CO2. Effectiveness of chx treatment in blocking new protein synthesis was verified by parallel experiments using [35S]methionine (data not shown). In experiments using dmj, cells were cultured overnight in medium containing 75 μg/ml dmj at 37 °C in 5% CO2; cell viability was identical in medium- and dmj-treated cultures (data not shown). Biotinylation of cell surface proteins was performed as described previously (24Wu W. Harley P.H. Punt J. Sharrow S.O. Kearse K.P. J. Exp. Med. 1996; 184: 759-794Crossref PubMed Scopus (51) Google Scholar). Cells were solubilized in 1% digitonin (Wako, Kyoto, Japan) lysis buffer (20 mm Tris, 150 mmNaCl, plus protease inhibitors) at 1 × 108 cells/ml for 20 min at 4 °C. Cell lysates were clarified by centrifugation to remove insoluble material and immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads as described previously (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Sequential immunoprecipitation, one- and two-dimensional SDS-PAGE gel electrophoresis, and immunoblotting were performed according to previously published methods (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). Recently we examined the reglucosylation of unassembled TCRα and -β proteins in BW thymoma cells using [3H]galactose as a radioactive tracer of Glc residues (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here we extended these studies in 2B4 T hybridoma cells to approximate at which stage(s) of TCR complex formation folding of individual TCR glycoproteins is completed, with the rationale that GT modification (reglucosylation) will cease upon attainment of proper conformation. As shown in Fig.1, [3H]galactose may be incorporated into N-linked oligosaccharides on glycoproteins via three major pathways: (i) conversion into UDP-[3H]galactose, the sugar donor for galactosyltransferase enzymes that transfer galactose residues to mature, complex-type oligosaccharides in the trans-Golgi; (ii) epimerization of UDP-[3H]galactose to UDP-[3H]glucose, the sugar donor for GT that transfers Glc residues to high mannose glycans on incompletely folded glycoproteins in the ER; and (iii) conversion of UDP-[3H]glucose into dolichol-phospho[3H]glucose, which is incorporated into nascent Glc3Man9GlcNAc2 glycans that are cotranslationally added to newly synthesized polypeptides in the ER (Fig. 1) (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hubbard S.C. Robbins P.W. J. Biol. Chem. 1979; 254: 4568-4576Abstract Full Text PDF PubMed Google Scholar, 27Suh K. Bergmann J.E. Gabel C.A. J. Cell Biol. 1989; 108: 811-819Crossref PubMed Scopus (57) Google Scholar, 28Varki A. Methods Enzymol. 1994; 230: 16-32Crossref PubMed Scopus (56) Google Scholar). In the current study, cycloheximide was included in all experiments to inhibit incorporation of [3H]glucose into newly translated proteins, thereby restricting radiolabeling to galactosylation and reglucosylation routes (Fig. 1) (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 2B4 T hybridoma cells were used, which have served as a model cell type for TCR assembly in numerous studies (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). As shown in Fig. 2 A, multiple TCR subunits were detected in anti-CD3ε precipitates of [3H]galactose-labeled 2B4 T cells, including CD3δ and -γ glycoproteins and clonotypic TCRα and -β proteins (Fig.2 A); as expected, nonglycosylated CD3ε and TCRζ molecules were not visualized (Fig. 2 A). Because anti-CD3ε precipitates contain a mixture of TCR components at various stages of their assembly superimposed upon one another, sequential precipitation techniques were used to separate more completely assembled TCR proteins (capable of becoming galactosylated in the Golgi) from partially assembled and unassembled TCR subunits (retained in the ER) (25Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). As demonstrated, when supernatants from anti-CD3ε precipitates were sequentially precipitated with anti-TCRβ mAb, radiolabeled TCRβ proteins were detected (Fig. 2 A), representing unassembled TCRβ proteins modified via the reglucosylation pathway. Consistent with incorporation of [3H]glucose into glycan chains on ER-localized TCRβ proteins, the radioactive signal on reglucosylated TCRβ proteins was sensitive to digestion with endoglycosidase H (EH), specific for immature oligosaccharides (data not shown). The vast majority of radiolabeled CD3γ glycoproteins associated with CD3ε were not simultaneously assembled with TCRβ but existed in partial complexes of CD3γε components (Fig. 2 A, anti-TCRβ → anti-CD3ε precipitates, respectively), indicating that most radiolabeled CD3γ chains associated with CD3ε were modified by GT. More than half of the CD3ε-associated CD3δ chains were assembled with TCRβ (Fig. 2 A), with remaining CD3δ proteins existing in partial δε complexes containing reglucosylatedN-glycans; unassembled CD3δ glycoproteins were also modified by GT as shown by sequential immunoprecipitation of anti-CD3ε precipitates with anti-CD3δ Ab to capture “free,” unassembled CD3δ chains (Fig. 2 A). Reglucosylation of partially assembled and free CD3γ and -δ glycoproteins was verified in 21.2.2 cells (Fig. 2 B), a TCRβ-deficient 2B4 variant that cannot assemble CD3 chains into a form capable of ER exit (29Bonifacino J.S. Chen C. Lippincott-Schwartz J. Ashwell J.D. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 35: 6929-6933Crossref Scopus (62) Google Scholar). Similar to our results in parental 2B4 cells, significantly more radiolabeled CD3γ proteins were associated with CD3ε than CD3δ proteins in 21.2.2 cells (Fig. 2 B). In agreement with previous studies showing that nascent glycoproteins undergo multiple cycles of deglucosylation and reglucosylation in the ER (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), Glc residues did not persist on CD3ε-associated γ proteins in 21.2.2 cells (Fig. 2 C), and CD3γ proteins were effectively radiolabeled during a secondary pulse period with [3H]galactose (Fig. 2 C). Taken together, these results demonstrate that glycosylated TCR subunits were substrates for GT, including invariant CD3γ and -δ molecules and clonotypic TCRβ proteins. In addition, these data show that both “free” and assembled CD3γ and -δ proteins contained reglucosylated glycans. To determine the contribution of reglucosylation in [3H]galactose radiolabeling of TCR proteins, studies were performed using the mannosidase inhibitor dmj, which precludes conversion of immature, high mannose glycans to mature (galactosylated) glycans in the Golgi (30Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar); thus, only reglucosylated glycoproteins are visualized in such experiments (Fig. 1). As shown in Fig.3 A, markedly fewer radiolabeled TCR proteins were present in anti-TCRα (A2B4) immunoprecipitates of dmj-treated cells relative to media-treated cells with TCRβ and CD3γ and -δ chains being completely absent and only TCRα proteins detected (Fig. 3 A). Consistent with restriction of radiolabeling to immature N-glycans, TCRα proteins in dmj lysates migrated with increased mobility compared with TCRα proteins from control lysates (Fig. 3 A) and unlike control TCRα molecules, disappeared completely following EH digestion (Fig. 3 A). Note that increased mobility of EH-digested TCRα-associated TCRβ proteins in control lysates results from the fact that several N-glycans on TCRβ proteins remain in the immature high mannose form, even on surface-expressed molecules, which is also true for CD3δ proteins (31, 32 and see below). Interestingly, increased amounts of reglucosylated (unassembled) TCRβ proteins existed in dmj lysates relative to control lysates (Fig.3 A), which was accompanied by augmented survival of newly synthesized TCRβ proteins. 2K. P. Kearse, unpublished observations. These results were specific in that the half-life and reglucosylation of unassembled TCRα molecules were relatively unaffected; similar results were observed in BW thymoma cells (data not shown). Biochemical analysis of surface-labeled molecules showed that the vast majority of TCR glycoproteins expressed on dmj-treated cells contained immature, EH-sensitive oligosaccharides (Fig. 3 B), demonstrating the effectiveness of dmj in blocking maturation ofN-oligosaccharides in these studies and showing that dmj treatment did not perturb TCR assembly. Taken together, these results show that most radiolabeled TCRβ and CD3γ and -δ glycoproteins associated with 2B4 TCRα represent galactosylated species and not reglucosylated TCR molecules. We conclude that TCRβ proteins assembled into TCRα and -β heterodimers are ineffectively modified by GT (and thus no longer perceived by the ER quality control system as incompletely folded) and relatedly, that reglucosylation of TCRβ and CD3γ and -δ proteins is terminated following their association with TCRα molecules. In addition, these data show that inhibition of mannosidase activity resulted in enhanced reglucosylation of unassembled TCRβ proteins. Assembly of TCRα and -β proteins into disulfide-linked heterodimers is preceded by the association of monomeric TCRα and TCRβ proteins with CD3 components in the form of αδε and βγε intermediates, which join to form αβδεγε complexes (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Thus, we next wished to determine whether CD3-associated TCRα and -β proteins were modified by GT. As shown in Fig. 4, both monomeric and dimeric radiolabeled TCRα and -β proteins were detected in association with CD3 chains in lysates of [3H]galactose-labeled 2B4 T cells (Fig. 4). Because monomeric TCRα and -β proteins are restricted to the ER (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar), these data indicate that reglucosylated (incompletely folded) TCRα and -β proteins exist in association with CD3 in 2B4 T cells, most likely as CD3α and CD3β intermediates because our previous results showed that few, if any, reglucosylated TCRβ proteins were associated with TCRα chains. To determine the contribution of GT modification to [3H]galactose radiolabeling of CD3-associated TCRα and -β proteins, dmj treatment was utilized to restrict radiolabeling to the reglucosylation pathway as before. In agreement with our previous findings that CD3γ and -δ glycoproteins assembled into partial δε, γε complexes were modified by GT, CD3γ and -δ chains were effectively labeled in dmj-treated cells (Fig.5 A). As noted earlier, reglucosylation of unassembled TCRβ chains, captured in sequential precipitates with anti-TCRβ mAb, was enhanced in dmj-treated cells relative to media-treated cells (Fig. 5 A). Importantly, these data show that reduced amounts of TCRα and -β proteins were associated with CD3 chains in dmj-treated cells compared with media-treated cells (Fig. 5 A), which was expected as our previous results showed that a significant portion of radiolabeled TCRα and -β proteins were assembled into disulfide-linked heterodimers modified by galactosylation. Analysis on two-dimensional nonreducing × reducing (NR × R) gels showed that relatively few radiolabeled TCRα and -β dimers were detected in anti-CD3 precipitates of dmj-treated cells (Fig. 5 B), unlike CD3 chains, which were readily visible (Fig. 5 B). Interestingly, radiolabeled TCRα proteins existed as both disulfide-linked and monomeric species whereas the vast majority of TCRβ molecules were present as non-disulfide-linked monomers (Fig. 5 B). Formation of TCRα and -β heterodimers was not precluded in dmj-treated cells as shown by immunoblotting of CD3 precipitates with anti-TCRα mAb (Fig. 6); dimeric TCRα proteins in media-treated groups existed as two species: an upper band representing mature (EH-resistant) proteins and a lower band migrating parallel with non-disulfide-linked TCRα monomers containing immature (EH-sensitive) glycans (Fig. 6). As demonstrated, only immature TCRα proteins were present in lysates of dmj-treated cells (Fig. 6). These results corroborate our previous findings that TCRβ subunits associated with TCRα proteins were ineffectively modified by GT and that reglucosylated (incompletely folded) CD3-associated monomeric TCRα and -β proteins exist in normal (untreated) cells. Taken together, these studies demonstrate that reglucosylation of most TCR components is extinguished following the CD3α and -β assembly and formation of disulfide-linked TCRα and -β heterodimers, indicating that TCR folding is finalized convergent with formation of αβδεγε complexes.Figure 5Reglucosylated TCR α and -β proteins are assembled with CD3 components in 2B4 T cells. A, 2B4 T cells were cultured in medium or dmj as indicated and labeled with [3H]galactose for 45 min. Digitonin lysates of equivalent numbers of cells were sequentially immunoprecipitated with 145-2C11 anti-CD3ε mAb, followed by H57-597 anti-TCRβ mAb. The positions of TCR proteins are marked.B, anti-CD3ε precipitates of dmj-treated [3H]galactose-labeled 2B4 cells shown in Awere analyzed on two-dimensional nonreducing x reducing (NR × R) gels. The positions of TCR proteins are marked.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Dimerization of TCR α proteins in media- and dmj-treated 2B4 T cells. Aliquots of the same cells used for [3H]galactose labeling in Fig. 5were lysed in digitonin and immunoprecipitated with 145-2C11 anti-CD3ε mAb. Lysates of equivalent numbers of cells were analyzed in media and dmj groups. Precipitates were analyzed on two-dimensional NR × R gels or digested with Endo H and analyzed on one-dimensional gels under reducing conditions and immunoblotted with anti-TCRα mAb. The positions of immature (EH-sensitive) and mature (EH-resistant) TCRα proteins are indicated; αD, dimeric α proteins; αM, monomeric α proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The current report has examined the modification of TCR glycoproteins by the ER folding sensor enzyme GT and provides the first example where GT modification of a multisubunit protein complex has been studied. The data in the current report significantly extend previous studies on TCR processing in splenic T lymphocytes, which showed that significant Glc trimming of newly synthesized CD3δ and TCRα glycoproteins takes place prior to association with partner TCR chains (33Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), based on their comigration with calnexin-associated glycoforms following digestion with jack bean α-mannosidase. Indeed, the current study utilizes a sensitive radiolabeling method which specifically identifies TCR subunits containing monoglucosylatedN-glycans generated via the reglucosylation pathway. The current report establishes that all TCR subunits containingN-glycans are substrates for GT and evaluated reglucosylation as a function of TCR assembly, previously examined only on unassembled TCRα and -β proteins expressed in BW thymoma cells, which do not efficiently assemble TCR complexes due to deficient CD3δ synthesis (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The results in this study suggest a scheme in which reglucosylation of invariant CD3γ and -δ subunits persists until their association with clonotypic TCRα and -β chains, and GT modification of TCRβ proteins is terminated following the assembly of αβδεγε complexes and formation of disulfide-linked TCRα and -β heterodimers. Interestingly, unlike CD3-associated TCRβ molecules, which were ineffectively modified by GT following disulfide linkage with TCRα proteins, reglucosylated TCRα molecules existed as both CD3-associated monomers and dimers. Although the exact significance of these findings remain to be determined, these data suggest that folding of TCRα may be one of the final steps of ER quality control that precedes TCR egress to the Golgi. It is conceivable that folding of the TCR complex occurs concomitant with the ordered assembly of TCR subunits and that GT recognition motifs become progressively “masked” as TCR oligomerization proceeds, similar to ER retention and lysosomal targeting information contained within the polypeptide sequences of certain TCR subunits (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar, 34Bonifacino J.S. Cosson P. Klausner R.D. Cell. 1990; 63: 503-513Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 35Letourner F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 36Dietrich J. Geisler C. J. Biol. Chem. 1998; 273: 26281-26284Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Consistent with this idea, the results in the current study provide evidence that most TCR glycoproteins are no longer perceived by the ER quality control system as incompletely folded following the assembly of αβδεγε TCR complexes, which, interestingly, is the stage at which TCR complexes become competent for ER exit. It is possible that reglucosylation of higher ordered TCR complexes ceases due to relocalization from the ER to the Golgi complex; however, we favor the idea that assembly, folding, and intracellular transport of TCR proteins are closely coupled events, similar to what has been observed for other multimeric immune protein complexes, i.e. major histocompatibility complex molecules (37Cresswell P. Cell. 1996; 84: 505-507Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 38Solheim J. Carreno B.M. Hansen T. J. Immunol. 1997; 158: 541-543PubMed Google Scholar, 39Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (874) Google Scholar). Indeed, previous studies have demonstrated that protein reglucosylation is not static but proceeds in a rapid, cyclic fashion in concert with Glc removal by glucosidase II enzymes (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar,16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Interestingly, we found that reglucosylation of “free” TCRβ proteins was increased under conditions of mannosidase blockade, which was specific in that GT modification of TCRα proteins was relatively unaffected.2 Because the efficiency of GT modification is inversely correlated with N-glycan chain length (12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and dmj inhibits the activity of certain ER mannosidase enzymes (40Weng S. Spiro R.G. J. Biol. Chem. 1993; 268: 25656-25663Abstract Full Text PDF PubMed Google Scholar,41Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar), it is reasonable that increased reglucosylation of unassembled TCRβ proteins in dmj-treated cells results from persistence of Man residues on N-glycan chains. However, it was also noted that the stability of newly synthesized TCRβ molecules was enhanced under these conditions, similar to what has been described for CD3δ proteins by Weissman and colleagues (6Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Thus, the relationship between increased reglucosylation and increased survival of TCR proteins under conditions of mannosidase blockade remains to be determined. Moreover, despite the fact that GT modification of certain TCR subunits was enhanced by prevention of Man removal, relatively few reglucosylated TCR proteins assembled into higher ordered TCR complexes were detected under these conditions, indicating that GT modification (folding) of TCR subunits is tightly regulated. Finally, it is unknown to what extent specific N-glycans on TCR proteins containing multiple oligosaccharides may be differentially modified by GT enzymes. Recent studies by Dessen et al. (42Dessen A. Gupta D. Sabewsan S. Brewer C.F. Sacchettini J.C. Biochemistry. 1995; 34: 4933-4942Crossref PubMed Scopus (152) Google Scholar) demonstrate that N-acetylglucosamine residues interact with neighboring amino acids of proteins in native conformations, which may be one of the major mechanisms by which GT modification of newly synthesized proteins is regulated (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar, 43Fanchiotti S. Fernandez F. Alessio C.D. Parodi A.J. J. Cell Biol. 1998; 143: 625-631Crossref PubMed Scopus (72) Google Scholar). The data in the current report suggest that determinants that signify malfolded molecules may persist on TCRα proteins compared with other TCR subunits, an idea that is consistent with previous findings that TCRα survival is uniquely sensitive to perturbations in the ER quality control system (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Suh K. Bergmann J.E. Gabel C.A. J. Cell Biol. 1989; 108: 811-819Crossref PubMed Scopus (57) Google Scholar, 44Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar). Identification of polypeptide and N-glycan domains important for GT recognition of TCR glycoproteins should provide valuable information regarding the molecular basis of GT modification and the regulation of quality control mechanisms that monitor the presence of unassembled and incompletely folded TCR proteins in the ER. We thank Drs. Velislava Karaivanova and Tom McConnell for critical reading of the manuscript. We are also grateful to Dr. Ralph Kubo for the gift of HMT3.2 Ab and Dr. Larry Samelson for the gift of R9 Ab.}, number={20}, journal={Journal of Biological Chemistry}, author={Gardner, T.G. and Kearse, K.P.}, year={1999}, pages={14094–14099} }
@article{gardner_kearse_1999, title={Purification of Immature CD4+CD8+ Thymocytes by Panning with Anti-CD8 Antibody}, volume={12}, DOI={10.1385/1-59259-682-7:47}, abstractNote={Most T lymphocytes of the immune system differentiate within the thymus along the CD4/CD8 developmental pathway by a highly ordered process termed thymic selection (,). The maturation status of thymocytes is commonly assessed by their expression of the coreceptor proteins CD4 and CD8 and their surface density of aβ T cell receptors (αβ TCR), (). Three major subpopulations of T cells exist within the thymus that exemplify the progression of thymocytes along the CD4/CD8 developmental pathway: 1. CD4−CD8− (double-negative) thymocytes which express no αβ TCR; 2. CD4+CD8+ (double-positive) thymocytes, which express no/low αβ TCR; and 3. CD4+CD8− and CD4−CD8+ (single positive) thymocytes, both of which express high surface density of αβ TCR (, , , ), (Fig. 1). Open image in new window Fig. 1. Intrathymic development of thymocytes along the CD4/CD8 developmental pathway. Immature CD4−CD8− thymocytes develop into progeny CD4+CD8+ thymocytes which maintain low expression of αβTCR. Immature CD4+CD8+ thymocytes expressing αβTCR of appropriate specificities are selected for further differentiation into mature T cells that express either CD4 (CD4+CD8−) or CD8 (CD4−CD8+) coreceptor molecules, but not both. Mature CD4+CD8− and CD4−CD8+ thymocytes emigrate from the thymus to the periphery where they may localize in lymphoid organs (for example, lymph node or spleen).}, journal={T Cell Protocols}, publisher={Humana Press}, author={Gardner, Terrence G. and Kearse, Kelly P.}, year={1999}, pages={47â53} }
@inproceedings{gardner_acosta-martinez_zobeck_baddock_pelt_senwo, title={Characterization of Microbes Carried in Dust}, DOI={10.13031/2013.39191}, abstractNote={There is still a lack of understanding of how soil microbial community distribution is controlled by wind erosion. This information is of international concern as eroded sediments can potentially carry away the active labile organic soil particulates containing key microorganisms involved in soil biogeochemical processes, which can have a negative impact on the quality and functional potential of the soil. Pyrosequencing techniques promises to expand our understanding of the vast microbial diversity with respect to soils that experience high rates of wind erosion; because it is able to sequence 10-100 times more DNA fragments than previous techniques (traditional cloning). Our study evaluated the bacterial diversity on coarse and fine dust collected from three different silty soils in Michigan by using a portable field wind tunnel instrument. Our results indicated that Acidobacter was the predominant bacteria in these soils as well as the predominant bacteria carried via wind dispersion in coarse and fine dust from these soil sources. Soil 1, which had higher P levels than the other 2 soils, pH was basically 6 and it had higher organic matter (OM) content (47.3-55%), while showing this order of bacterial predominance: Acidobacter, Streptomyces, Levilinea, Patulibacter and Gemmatimonas. Although Streptomyces was the second most abundant bacteria in soil source 1, fine dust did not carry this species, and Levilinea was the second most predominant bacteria in this dust. Soil 2, which had lower P levels than soil 1 (within a range of 122-136 mg P kg-1), pH of about 5.5, and an intermediate OM content (42.9%) also showed the species predominance of Acidobacter followed by Patulibacter, Conexibacter, Rhizobium and Levilinea. Three of the 5 predominant bacteria in the soil source were also predominant in the fine dust except for Conexibacter and Rhizobium. Soil 3 had the lowest OM content (16.3-20.8%) of the 3 soils evaluated, and it had an average pH of 5.7, and P levels within a range of 123-153 P mg kg-1. This soil also showed a predominance of Acidobacter followed by Patulibacter, Rhizobium, Gemmatimonas, and Conexibacter. In addition to Acidobacter in fine dust, Conexibacter and Patulibacter were also carried. The coarse dust samples collected from these 3 soils demonstrated some differences in bacterial distribution compared to the fine dust, which may indicate that fine dust dispersion caused by wind erosion is the major carrier of soil predominant bacteria. The highest abundance of Acidobacter is explained by the acidic pH of these soils, and thus, it appears that they play an important ecological role in these soils functioning. Our findings suggested that bacteria carried in coarse or fine dust represent fingerprints of the soil source, but certain specific group of bacteria was more abundant in fine dust than coarse dust, revealing different niches in these soils. We are in the process of identifying the ecological role of the bacterial groups carried in these dust samples as they can have important implications on the soil sustainability and functioning. This research is focused on coupling the diversity of the soil microbial communities carried by wind erosion with biogeochemical functionality using enzymatic activities involved in nutrient cycling. This will allow us to identify keystone microbial species-assemblages associated with biogeochemical processes of the soil source.}, booktitle={International Symposium on Erosion and Landscape Evolution (ISELE), 18-21 September 2011, Anchorage, Alaska}, publisher={American Society of Agricultural and Biological Engineers}, author={Gardner, Terrence G and Acosta-Martinez, Veronica and Zobeck, Ted and Baddock, Matthew C and Pelt, Robert Scott Van and Senwo, Zachary} }