Prolonged Amphetamine Treatments Cause Long-Term Decrease of Dopamine Uptake in Cultured Cells

Neurochemical Research - Amphetamine (AMPH) is a systemic stimulant used to treat a variety of diseases including Attention Deficit Hyperactive Disorder, narcolepsy and obesity. Previous data...     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

Electrical stimulation of human embryonic stem cells: Cardiac differentiation and the generation of reactive oxygen species

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H₂O₂. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.     

EGFR through STAT3 modulates ΔN63α expression to sustain tumor‐initiating cell proliferation in squamous cell carcinomas

Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR–ΔNp63α axis in proliferation of SCC tumor‐initiating cells (TICs). SCC cell lines A‐431, Cal‐27, and SCC‐25 treated with EGF showed a time‐dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non‐differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non‐differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR–ΔNp63α axis is crucial for proliferation of TICs present in SCCs. J. Cell. Physiol. 228: 871–878, 2013. © 2012 Wiley Periodicals, Inc.     

Loss of genetic integrity and biological invasions result from stocking and introductions of Barbus barbus: insights from rivers in England

Anthropogenic activities, including the intentional releases of fish for enhancing populations (stocking), are recognized as adversely impacting the adaptive potential of wild populations. Here, the genetic characteristics of European barbel Barbus barbus were investigated using 18 populations in England, where it is indigenous to eastern‐flowing rivers and where stocking has been used to enhance these populations. Invasive populations are also present in western‐flowing rivers following introductions of translocated fish. Two genetic clusters were evident in the indigenous range, centered on catchments in northeast and southeast England. However, stocking activities, including the release of hatchery‐reared fish, have significantly reduced the genetic differentiation across the majority of this range. In addition, in smaller indigenous rivers, populations appeared to mainly comprise fish of hatchery origin. In the nonindigenous range, genetic data largely aligned to historical stocking records, corroborating information that one particular river (Kennet) in southeast England was the original source of most invasive B. barbus in England. It is recommended that these genetic outputs inform management measures to either restore or maintain the original genetic diversity of the indigenous rivers, as this should help ensure populations can maintain their ability to adapt to changing environmental conditions. Where stocking is considered necessary, it is recommended that only broodstock from within the catchment is used.     

Hypothermia during Carotid Endarterectomy: A Safety Study

BackgroundCEA is associated with peri-operative risk of brain ischemia, due both to emboli production caused by manipulation of the plaque and to potentially noxious reduction of cerebral blood flow by carotid clamping. Mild hypothermia (34–35°C) is probably the most effective approach to protect brain from ischemic insult. It is therefore a substantial hypothesis that hypothermia lowers the risk of ischemic brain damage potentially associated with CEA. Purpose of the study is to test whether systemic endovascular cooling to a target of 34.5–35°C, initiated before and maintained during CEA, is feasible and safe.MethodsThe study was carried out in 7 consecutive patients referred to the Vascular Surgery Unit and judged eligible for CEA. Cooling was initiated 60–90 min before CEA, by endovascular approach (Zoll system). The target temperature was maintained during CEA, followed by passive, controlled rewarming (0.4°C/h). The whole procedure was carried out under anesthesia.ResultsAll the patients enrolled had no adverse events. Two patients exhibited a transient bradycardia (heart rate 30 beats/min). There were no significant differences in the clinical status, laboratory and physiological data measured before and after CEA.ConclusionsSystemic cooling to 34.5–35.0°C, initiated before and maintained during carotid clamping, is feasible and safe.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02629653","term_id":"NCT02629653"}}NCT02629653     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Synthesis and enantioselectivity of the enantiomers of PG9 and SM21, new potent analgesic and cognition-enhancing drugs

The enantiomers of two α-tropanyl esters, SM₂₁ (1) and PG₉ (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(−)-S-1, (−)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM₂₁, (+)-S-PG₉]. The ACh-releasing effect of the enantiomers of SM₂₁ in rats is in agreement with the results in mice, while PG₉ enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT₄ antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the α-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds. © 1996 Wiley-Liss, Inc.     

The application of a plug-flow reactor to measure the biodegradable dissolved organic carbon (BDOC) in seawater

Most of the ambient dissolved organic carbon (DOC) is refractory to microbial degradation; bacteria can consume a minor but variable part of the DOC pool over periods of hours and days. It is important to increase our knowledge of the dynamics of the biodegradable fraction of DOC (BDOC) to understand the global carbon budget.Several methods for determining BDOC have been developed; however, the problem of most of them is the time (days/weeks) required for the colonization and/or determination.In this paper, we describe the application to seawater of a plug-flow bioreactor to measure BDOC within 3–4 h. The bioreactor was built following Søndergaard and Worm [Søndergaard, M., Worm, J., 2001. Measurement of biodegradable dissolved organic carbon (BDOC) in lake water with a bioreactor. Water Res. 35, 2505-2513.] protocols for the measurement of BDOC in lake water. We analyzed BDOC on samples collected in the Gulf of Trieste during autumn–winter and summer 2003–2004. BDOC concentrations varied from 8 to 24 μM and represented from 10.3% to 25.5% of the total DOC. To evaluate the effectiveness of this method, we compared bioreactor BDOC measurement with data obtained from batch cultures. The results indicate that BDOC in coastal seawater can be measured rapidly and reliably with this bioreactor.