Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Draft genome sequence of Leucobacter chromiiresistens, an extremely chromium-tolerant strain

Here we present the draft genome of Leucobacter chromiiresistens. This is the first genome sequence of an organism belonging to the genus Leucobacter. L. chromiiresistens was sequenced due to its capability to tolerate up to 300 mM Cr(VI) in the medium, which is so far a unique feature for microorganisms.     

Superior Therapeutic Index of Calmangafodipir in Comparison to Mangafodipir as a Chemotherapy Adjunct

Mangafodipir is a magnetic resonance imaging contrast agent with manganese superoxide dismutase (MnSOD) mimetic activity. The MnSOD mimetic activity protects healthy cells against oxidative stress-induced detrimental effects, e.g., myelosuppressive effects of chemotherapy drugs. The contrast property depends on in vivo dissociation of Mn²⁺ from mangafodipir—about 80% dissociates after injection. The SOD mimetic activity, however, depends on the intact Mn complex. Complexed Mn²⁺ is readily excreted in the urine, whereas dissociated Mn²⁺ is excreted slowly via the biliary route. Mn is an essential but also a potentially neurotoxic metal. For more frequent therapeutic use, neurotoxicity due to Mn accumulation in the brain may represent a serious problem. Replacement of 4/[5] of Mn²⁺ in mangafodipir with Ca²⁺ (resulting in calmangafodipir) stabilizes it from releasing Mn²⁺ after administration, which roughly doubles renal excretion of Mn. A considerable part of Mn²⁺ release from mangafodipir is governed by the presence of a limited amount of plasma zinc (Zn²⁺). Zn²⁺ has roughly 10³ and 10⁹ times higher affinity than Mn²⁺ and Ca²⁺, respectively, for fodipir. Replacement of 80% of Mn²⁺ with Ca²⁺ is enough for binding a considerable amount of the readily available plasma Zn²⁺, resulting in considerably less Mn²⁺ release and retention in the brain and other organs. At equivalent Mn²⁺ doses, calmangafodipir was significantly more efficacious than mangafodipir to protect BALB/c mice against myelosuppressive effects of the chemotherapy drug oxaliplatin. Calmangafodipir did not interfere negatively with the antitumor activity of oxaliplatin in CT2[6] tumor-bearing syngenic BALB/c mice, contrary calmangafodipir increased the antitumor activity.     

Accelerating regrowth of temperate‐maritime forests due to environmental change

To understand how environmental changes have influenced forest productivity, stemwood biomass (B) dynamics were analyzed at 1267 permanent inventory plots, covering a combined 209 ha area of unmanaged temperate‐maritime forest in southwest British Columbia, Canada. Net stemwood production (ΔB) was derived from periodic remeasurements of B collected over a 40‐year measurement period (1959–1998) in stands ranging from 20 to 150 years old. Comparison between the integrated age response of net stemwood production, ΔB(A), and the age response of stemwood biomass, B(A), suggested a 58 ± 11% increase in ΔB between the first 40 years of the chronosequence period (1859–1898) and the measurement period. To estimate extrinsic forcing on ΔB, several different candidate models were developed to remove variation explained by intrinsic factors. All models exhibited temporal bias, with positive trends in (observed minus predicted) residual ΔB ranging between of 0.40 and 0.64% yr^?1. Applying the same methods to stemwood growth (G) indicated residual increases ranging from 0.43 and 0.67% yr^?1. Higher trend estimates corresponded with models that included site index (SI) as a predictor, which may reflect exaggeration of the age‐decline in SI tables. Choosing a model that excluded SI, suggested that ΔB increased by 0.40 ± 0.18% yr^?1, while G increased by 0.43 ± 0.12% yr^?1 over the measurement period. Residual G was significantly correlated with atmospheric carbon dioxide (CO₂), temperature (T), and climate moisture index (CMI). However, models driven with climate and CO₂, alone, could not simultaneously explain long‐term and measurement‐period trends without additional representation of indirect effects, perhaps reflecting compound interest on direct physiological responses to environmental change. Evidence of accelerating forest regrowth highlights the value of permanent inventories to detect and understand systematic changes in forest productivity caused by environmental change.     

Results of the clinical donor case and quality system case workshops of the European Association of Tissue Banks annual meeting 2009

The European Association of Tissue Banks (EATB) Donor Case Workshop and Quality System Case workshop are forums held within the program of the EATB Annual Congress. These workshops offer an opportunity to discuss and evaluate approaches taken to challenging situations, regarding donor selection and quality issues, and strengthen the professional tissue banking and regulatory networks across Europe. This report reflects some of the discussion at the congress workshops and also subsequent correspondence between the various individuals who submitted cases for discussion. The cases presented to the workshops demonstrate that the findings, their interpretation, deducted actions and preventive measures in tissue banks are not predictable. The varied responses and lack of consensus corroborate this and clearly indicate that operating procedures cannot comprehensively cover or prepare for all eventualities. For many of the issues raised there is a lack of information in the published literature. The workshops actively engage participants, representing a wide array of international expertise, in an informal, secure and enjoyable setting, which facilitates learning from peers and provides potential solutions to those submitting cases. By publishing a summary of the discussions, we hope to reach a wider audience and to stimulate individuals to undertake full literature reviews or research on some of the discussed subjects.     

Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation

Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P(2)Y(12), P(2)Y(1) and PI3K p110β completely inhibits hypothermic ECC-induced activation processes. This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.     

The penguin waddling gait pattern has a more consistent step width than step length

Previous research has indicated that the sagittal plane gait dynamics of humans are more stable and less dependent on active neural control, while the frontal plane dynamics are less stable and require greater neural control. The higher neural demands of the frontal plane dynamics are reflected in a more variable step width than step length. Greater variability in the step width occurs because humans modulate their foot placement for each step to ensure stability and prevent falls. Compared to other terrestrial animals, penguins appear to have excessive amount of frontal plane motion in their gait that is characterized as waddling. If excessive frontal plane motion requires additional neural control and is associated with falls, it would seem that evolutionary pressures would have eliminated such locomotive strategies. Here we measured the step length and width variability to determine if waddling results in a less stable gait. Remarkably, the variability of the step width was less than the variability of the step length. These results are directly opposite of what has been reported for humans. Hence, our data indicate that waddling may be an effective strategy for ensuring stability in the frontal plane dynamics.     

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.     

Lysosomes in iron metabolism,ageing and apoptosis

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ∼4 to 5) that constantly fuse and divide. It receives a large number of hydrolases (∼50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.