Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.     

A TNF receptor 2 selective agonist rescues human neurons from oxidative stress-induced cell death

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.     

Use-dependent plasticity in barrel cortex: intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns

We used optical imaging of intrinsic cortical signals, elicited by whisker stimulation, to define areas of activation in primary sensory cortex of normal hamsters and hamsters subjected to neonatal follicle ablation at postnatal day seven (P7). Follicle ablations were unilateral, and spared either C-row whiskers or the second whisker arc. This study was done to determine if the intrinsic cortical connectivity pattern of the barrel cortex, established during the critical period, affects the process of representational plasticity that follows whisker follicle ablation. Additionally, we tested the ability to monitor such changes in individual cortical whisker representations using intrinsic signal imaging. Stimulation of a single whisker yielded peak activation of a barrel-sized patch in the somatotopically appropriate location in normal cortex. In both row and arc-spared animals, functional representations corresponding to spared follicles were significantly stronger and more oblong than normal. The pattern of activation differed in the row-sparing and arc-sparing groups, in that the expansion was preferentially into deprived, not spared areas. Single whisker stimulation in row-spared cases preferentially activated the corresponding barrel arc, while stimulation of one whisker in arc-spared cases produced elongated activation down the barrel row. Since whisker deflection normally has a net inhibitory effect on neighboring barrels, our data suggest that intracortical inhibition fails to develop normally in deprived cortical columns. Because thalamocortical projections are not affected by follicle ablation after P7, we suggest that the effects we observed are largely cortical, not thalamocortical.     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.     

Nickel accumulation and storage in Bradyrhizobium japonicum

Hydrogenase-derepressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl2, and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown and derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunoprecipitation of 63Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms.     

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.     

The effect of temperature on the development times of eggs,naupliar and copepodite stages of five species of cyclopoid copepods

The duration times of eggs, combined naupliar instars and of the different copepodite stages of five species of cyclopoid copepods — Acanthocyclops robustus, Cyclops vicinus, Diacyclops bicuspidatus, Mesocyclops leuckarti, and Thermocyclops crassus — were investigated at five different temperatures. The five species can be divided in two groups: two species, C. vicinus and D. bicuspidatus, adapted to cold water conditions and three species, A. robustus, M. leuckarti and T. crassus adapted to warm water conditions. The cold water species showed a faster egg development than M. leuckarti and T. crassus at 5–15 °C. The eggs and instars of the warm water species M. leuckarti tend to develop faster than those of the former two species at higher temperatures. A. robustus showed the shortest egg and instar development at 10–25 °C. The warm water species T. crassus produced no eggs at 10 °C and temperatures below. At higher temperatures (20, 25 °C) the egg and instar duration times were similar or longer than those of the other species. When cultured in total darkness a great part of the C_IV respectively C_V copepodites of the summer forms entered arrest and the percentage of copepodites that showed an arrest of development was highest at lowest temperatures. The present results are compared with data from literature and differences are discussed.