The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand-LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.     

Asymmetric localization of calpain 2 during neutrophil chemotaxis

Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.     

Natural Monoterpenes: Much More than Only a Scent

Terpenes are a widespread group of secondary metabolites that can be found in various family plants such as the Lamiaceae. In view of their numerous valuable biological activities, the industrial production of concrete terpenes and essential oils rich in the substances is intensively studied. Monoterpenes constitute a significant part of the whole group of the aforementioned secondary metabolites. This is due to their numerous biological activities and their ability to permeate the skin. Despite the fact that these substances have gain popularity, they are not comprehensively characterized. The presented review is based on studies of the biological activities of the most important monoterpenes and the essential oils rich in these compounds. The authors focused attention on antioxidant activity, inhibition towards acetyl‐ and butyrylcholinesterase, and α‐amylase and α‐glucosidase, antifungal, hepatoprotective, sedative properties, and their skin permeation enhancement.     

Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.     

Sowing of margin strips rich in floral resources improves herbivore control in adjacent crop fields

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Modelling time‐varying low‐temperature‐induced mortality rates for pupae of Tuta absoluta (Gelechiidae,Lepidoptera)

In a series of laboratory experiments, acclimated pupae of Tuta absoluta were exposed to various constant low temperatures in order to estimate their maximum survival times (Kaplan–Meier, Lt_99.99). A Weibull function was fitted to the data points, describing maximum survival time as a function of temperature. In another experiment at ?6°C, the progress of mortality increasing with exposure time was identified. These values were fitted by a sigmoidal function converging asymptotically to 100% mortality for very long exposure times. Analysing mortality data from the maximum survival experiment by a generalized linear model showed a significant common slope parameter (p < .001) that reveals parallelism of the survival curves at each temperature if a log time axis is used. These curves appear stretched (time scaled) if plotted with a nonlogarithmic time axis. By combining these mathematical relations, it was possible to calculate a species‐specific ‘mortality surface’ which exhibits mortalities, depending on temperature and duration of exposure. In order to accumulate hourly mortalities for courses of varying temperatures, an algorithm was developed which yields mortality values from that surface taking into account the attained mortality level. In validation experiments, recorded mortalities were compared against modelled mortalities. Prediction of mortality was partially supported by the model, but pupae experiencing intensely fluctuating temperatures showed decreased mortality, probably caused by rapid cold hardening during exposure. Despite this observation, mortality data converged to distinct levels very close to 100% depending on the intensity of temperature fluctuations that were characteristic for different types of experiments. The highest mortality limit occurred at intensely fluctuating temperatures in laboratory experiments. This constituted a benchmark that was not reached under various field conditions. Thus, it was possible to identify temperature limits for the extinction of field populations of Tuta absoluta pupae.     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.