Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics

Protein glycosylation is an important post‐translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N‐glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody‐dependent cell‐mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1419–1431, 2014     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.     

Global synthesis of the temperature sensitivity of leaf litter breakdown in streams and rivers

Streams and rivers are important conduits of terrestrially derived carbon (C) to atmospheric and marine reservoirs. Leaf litter breakdown rates are expected to increase as water temperatures rise in response to climate change. The magnitude of increase in breakdown rates is uncertain, given differences in litter quality and microbial and detritivore community responses to temperature, factors that can influence the apparent temperature sensitivity of breakdown and the relative proportion of C lost to the atmosphere vs. stored or transported downstream. Here, we synthesized 1025 records of litter breakdown in streams and rivers to quantify its temperature sensitivity, as measured by the activation energy (E_a, in eV). Temperature sensitivity of litter breakdown varied among twelve plant genera for which E_a could be calculated. Higher values of E_a were correlated with lower‐quality litter, but these correlations were influenced by a single, N‐fixing genus (Alnus). E_a values converged when genera were classified into three breakdown rate categories, potentially due to continual water availability in streams and rivers modulating the influence of leaf chemistry on breakdown. Across all data representing 85 plant genera, the E_a was 0.34 ± 0.04 eV, or approximately half the value (0.65 eV) predicted by metabolic theory. Our results indicate that average breakdown rates may increase by 5–21% with a 1–4 °C rise in water temperature, rather than a 10–45% increase expected, according to metabolic theory. Differential warming of tropical and temperate biomes could result in a similar proportional increase in breakdown rates, despite variation in E_a values for these regions (0.75 ± 0.13 eV and 0.27 ± 0.05 eV, respectively). The relative proportions of gaseous C loss and organic matter transport downstream should not change with rising temperature given that E_a values for breakdown mediated by microbes alone and microbes plus detritivores were similar at the global scale.     

New insights into microbial oxidation of antimony and arsenic

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.     

Two imide substances from a soil-isolated Streptomyces atratus strain provide effective biocontrol activity against grapevine downy mildew

Grapevine downy mildew (Plasmopara viticola) is a devastating disease of grapevine. In this study, 151 actinomycete isolates were obtained and tested for antagonistic activity against P. viticola. The assay suggested that 28 isolates displayed antagonistic effects to varying degrees. The greatest reduction in disease severity was observed with isolate PY-1, which reduced disease severity by 92.13% in the detached leaf assay, and by 83% in a field assay. It was identified as Streptomyces atratus using the 16S rDNA sequence analysis. To elucidate the antagonistic mechanism of PY-1 against P. viticola, scanning electron microscopy showed that major damage to the pathogens sporangia and sporangiophores was observed after treatment for PY-1. Furthermore, PY-1 showed antagonistic activity against other pathogens, including: Botrytis cinerea, Colletotrichum orbiculare, Fusarium oxysporum, Phytophthora capsici and Phytophthora infestans. Two imide compounds were purified from the fermentation liquid using silica gel chromatography and high-performance liquid chromatography and identified as 5-acetoxycycloheximide and cycloheximide using nuclear magnetic resonance. Both compounds showed significant antagonistic activity against P. viticola, determining a reduction in disease severity by 65% and 84%, respectively. In conclusion, 5-acetoxycycloheximide and cycloheximide were identified for the first time in a new S. atratus strain able to effectively control grapevine downy mildew.