Gaseous nitrogen losses from field plots grown with pea (Pisum sativum L.) or spring barley (Hordeum vulgare L.) estimated by 15N mass balance and acetylene inhibition techniques

In a mass balance of ¹⁵N-labelled nitrate added to soil grown with pea or barley, denitrification estimates using the acetylene-inhibition technique were compared with unaccounted for ¹⁵N. During the growth season of 1989, which was drier than average, N losses due to denitrification estimated by the acetylene-inhibition technique were negligible. A substantial amount of fertilizer N was unaccounted for by the ¹⁵N mass balance, especially in the pea plots. The loss took place during the period of grain-filling in which no leaching occurred, and was accompanied by a decrease in ¹⁵N content of the plants. Volatilization of ammonia from the aerial parts of the plants is a possible explanation of the observed loss. An estimation of denitrification relying only on the ¹⁵N mass balance would have resulted in an overestimation of denitrification.     

Effervescence and Ephemerality: Popular Urban Uprisings in Mozambique

This article analyses the large-scale popular urban uprisings that shook Mozambican cities on 1 and 2 September 2010, following the government's announcement of successive rises in the price of public transport fares and basic commodities. Using ethnographic material from the city of Chimoio and the capital Maputo, the following work highlights the organisational character of the ‘strikes’ (greves), as the popular uprisings were called, and explores them as a new form of organising political discontent. Comparing them to other historical and contemporary popular uprisings, this article argues that the strikes violently and rhizomically generated ephemeral and egalitarian forms of political authority and order that simultaneously confronted, replicated and undercut the aspects of Mozambican statehood. Deploying Durkheim's notion of effervescence, the work further argues that the creative fervour, multisemic aspects and festive character of the popular uprisings need to be recognised; thus, this analysis challenges the reductive labelling of these events as ‘riots’ or ‘food riots’.     

A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.     

A Combretastatin-Mediated Decrease in Neutrophil Concentration in Peripheral Blood and the Impact on the Anti-Tumor Activity of This Drug in Two Different Murine Tumor Models

The vascular disrupting agent combretastatin A-4 disodium phosphate (CA4P) induces fluctuations in peripheral blood neutrophil concentration. Because neutrophils have the potential to induce both vascular damage and angiogenesis we analyzed neutrophil involvement in the anti-tumoral effects of CA4P in C3H mammary carcinomas in CDF1 mice and in SCCVII squamous cell carcinomas in C3H/HeN mice. Flow cytometry analyses of peripheral blood before and up to 144 h after CA4P administration (25 and 250 mg/kg) revealed a decrease 1 h after treatment, followed by an early (3–6 h) and a late (>72 h) increase in the granulocyte concentration. We suggest that the early increase (3–6 h) in granulocyte concentration was caused by the initial decrease at 1 h and found that the late increase was associated with tumor size, and hence independent of CA4P. No alterations in neutrophil infiltration into the C3H tumor after CA4P treatment (25 and 250 mg/kg) were found. Correspondingly, neutrophil depletion in vivo, using an anti-neutrophil antibody, followed by CA4P treatment (25 mg/kg) did not increase the necrotic fraction in C3H tumors significantly. However, by increasing the CA4P dose to 250 mg/kg we found a significant increase of 359% in necrotic fraction when compared to neutrophil-depleted mice; in mice with no neutrophil depletion CA4P induced an 89% change indicating that the presence of neutrophils reduced the effect of CA4P. In contrast, neither CA4P nor 1A8 affected the necrotic fraction in the SCCVII tumors significantly. Hence, we suggest that the initial decrease in granulocyte concentration was caused by non-tumor-specific recruitment of neutrophils and that neutrophils may attenuate CA4P-mediated anti-tumor effect in some tumor models.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

Within-season flowering interruptions are common in the water-limited Sky Islands

Within-season breaks in flowering have been reported in a wide range of highly variable ecosystems including deserts, tropical forests and high-elevation meadows. A tendency for interruptions in flowering has also been documented in southwestern US “Sky Island” plant communities, which encompass xeric to mesic conditions. Seasonal breaks in flowering have implications for plant reproductive success, population structure, and gene flow as well as resource availability for pollinators and dependent animals. Most reports of multiple within-season flowering events describe only two distinct flowering episodes. In this study, we set out to better quantify distinct within-season flowering events in highly variable Sky Islands plant communities. Across a >1,200 m elevation gradient, we documented a strong tendency for multiple within-season flowering events. In both distinct spring and summer seasons, we observed greater than two distinct within-season flowering in more than 10 % of instances. Patterns were clearly mediated by the different climate factors at work in the two seasons. The spring season, which is influenced by both temperature and precipitation, showed a mixed response, with the greatest tendency for multiple flowering events occurring at mid-elevations and functional types varying in their responses across the gradient. In the summer season, during which flowering across the gradient is limited by localized precipitation, annual plants exhibited the fewest within-season flowering events and herbaceous perennial plants showed the greatest. Additionally, more distinct events occurred at lower elevations. The patterns documented here provide a baseline for comparison of system responses to changing climate conditions.     

A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast

A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.     

Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis

A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information on the residual secondary structure elements in the acid-denatured state of an acyl-coenzyme A binding protein. This method reveals a clear correlation between the carbon secondary chemical shifts and the amide secondary chemical shifts 3-5 residues away in the primary sequence. These findings strongly suggest transient formation of short helix-like segments, and identify unique sequence segments important for protein folding.     

Effect of protein-surfactant interactions on aggregation of β-lactoglobulin

The milk protein β-lactoglobulin (βLG) dominates the properties of whey aggregates in food products. Here we use spectroscopic and calorimetric techniques to elucidate how anionic, cationic and non-ionic surfactants interact with bovine βLG and modulate its heat-induced aggregation. Alkyl trimethyl ammonium chlorides (xTAC) strongly promote aggregation, while sodium alkyl sulfates (SxS) and alkyl maltopyranosides (xM) reduce aggregation. Sodium dodecyl sulfate (SDS) binds to non-aggregated βLG in several steps, but reduction of aggregation was associated with the first binding step, which occurs far below the critical micelle concentration. In contrast, micellar concentrations of xMs are required to reduce aggregation. The ranking order for reduction of aggregation (normalized to their tendency to self-associate) was C10-C12>C8>C14 for SxS and C8>C10>C12>C14>C16 for xM. xTAC promote aggregation in the same ranking order as xM reduce it. We conclude that SxS reduce aggregation by stabilizing the protein's ligand-bound state (the melting temperature t(m) increases by up to 10°C) and altering its charge potential. xM monomers also stabilize the protein's ligand-bound state (increasing t(m) up to 6°C) but in the absence of charged head groups this is not sufficient by itself to prevent aggregation. Although micelles of both anionic and non-ionic surfactants destabilize βLG, they also solubilize unfolded protein monomers, leaving them unavailable for protein-protein association and thus inhibiting aggregation. Cationic surfactants promote aggregation by a combination of destabilization and charge neutralization. The food compatible surfactant sodium dodecanoate also inhibited aggregation well below the cmc, suggesting that surfactants may be a practical way to modulate whey protein properties.