Comparative effects of sodium pyrithione evoked intracellular calcium elevation in rodent and primate ventral horn motor neurons

Oral administration of sodium pyrithione (NaP) causes hindlimb weakness in rodents, but not in primates. Previous work using Aplysia neurons has demonstrated that NaP produces a persistent influx of Ca²⁺ ions across the plasma membrane. To determine whether this also occurs in mammalian neurons and whether this could underlie the inter-species difference between rodents and primates, we have tested the effects of NaP on intracellular Ca²⁺ levels ([Ca²⁺]_i) in rat and monkey motor neurons in vitro. Motor neurons present in spinal cord slices from rhesus monkey embryos (E37 and 56) and from rat E16 were dissected and cultured on glass coverslips. Following 2 weeks (rhesus) or 2-3 days (rat) in culture, neurons were loaded with fura-PE3/AM, and examined for [Ca²⁺]_i changes in response to NaP. Rhesus motor neurons were identified by immunostaining for Islet-1 (MN specific antigen) and neuron specific enolase (NSE). Motor neurons from both species exhibited dose-dependent NaP-evoked increases in [Ca²⁺]_i However, the dose-response curve for the Rhesus motor neurons was significantly shifted to the right of the rat dose-response curve, whereas the overall amplitude of the Ca²⁺ rise was similar in both species. As shown previously for the Aplysia neurons, the action of NaP is attenuated by SKF 96365, an inhibitor of store-operated calcium entry. In contrast the action of NaP is unaffected by nifedipine and tetrodotoxin, blockers of voltage-dependent Ca²⁺ and Na⁺ channels, respectively, or by ouabain, an inhibitor of the plasma membrane Na⁺/K⁺ ATPase. Our results indicate that the NaP-induced increase in [Ca²⁺]_i is conserved across species and suggest that the toxicological sensitivity of rodent over primate to pyrithione could be due to the enhanced sensitivity of rodent motor neurons to NaP-evoked intracellular Ca²⁺ elevation.     

Simultaneous determination of doxifluridine and 5-fluorouracil in monkey serum by high performance liquid chromatography with tandem mass spectrometry

A reverse-phase high performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its active metabolite 5-fluorouracil in monkey serum. A liquid/liquid extraction with ethyl acetate (90%) and isopropyl alcohol (10%) was used to extract simultaneously doxifluridine and 5-FU which have considerable difference in the polarity. Optimum chromatographic separation was achieved on a Agilent Zorbax C(18) (100 mm x 2.1mm, 3.5 microm) column with a mobile phase of methanol-water (20:80, v/v). The flow rate was 0.2 mL/min with total cycle time of 5 min. The lower limit of quantification (LLOQ) was validated at 10.0 ng/mL of serum for both doxifluridine and 5-FU. Accuracy and precision of quality control (QC) samples for both compounds met FDA Guidance criteria of +/-15% with average QC accuracy of 95.5-105.0% and coefficients of variation of 1.1-9.5% in the 10-2000 ng/mL concentration range. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability to support the analysis of monkey serum samples.     

Atrial fibrillation is not uncommon among patients with ischemic stroke and transient ischemic stroke in China

Background

Atrial fibrillation (AF) is reported to be a less frequent cause of ischemic stroke in China than in Europe and North America, but it is not clear whether this is due to underestimation. Our aim was to define the true frequency of AF-associated stroke, to determine the yield of 6-day Holter ECG to detect AF in Chinese stroke patients, and to elucidate predictors of newly detected AF.

Methods

Patients with acute ischemic stroke or transient ischemic attack (TIA) were enrolled in a prospective, multicenter cohort study of 6-day Holter monitoring within 7 days after stroke onset at 20 sites in China between 2013 and 2015. Independent predictors of newly-detected AF were determined by multivariate analysis.

Results

Among 1511 patients with ischemic stroke and TIA (mean age 63 years, 33.1% women), 305 (20.2%) had either previously known (196, 13.0%) or AF newly-detected by electrocardiography (53, 3.5%) or by 6-day Holter monitoring (56/1262, 4.4%). A history of heart failure (OR?=?4.70, 95%CI, 1.64–13.5), advanced age (OR?=?1.06, 95%CI, 1.04–1.09), NIHSS at admission (OR?=?1.06, 95%CI, 1.02–1.10), blood high density lipoprotein (HDL) (OR?=?1.52, 95%CI, 1.09–2.13), together with blood triglycerides (OR?=?0.64, 95%CI, 0.45–0.91) were independently associated with newly-detected AF.

Conclusions

Contrary to previous reports, AF-associated stroke is frequent (20%) in China if systemically sought. Prolonged noninvasive cardiac rhythm monitoring importantly increases AF detection in patients with recent ischemic stroke and TIA in China. Advanced age, history of heart failure, and higher admission NIHSS and higher level of HDL were independent indicators of newly-detected AF.

Trial registration

NCT02156765 (June 5, 2014).

     

Focal adhesion kinase maintains,but not increases the adhesion of dental pulp cells

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.     

The hyperthermophilic α-amylase from <Emphasis Type="Italic">Thermococcus</Emphasis> sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.     

Transient myeloproliferative disorder in a newborn with down syndrome treated with rasburicase for the risk of development of tumor lysis syndrome: A case report

Introduction

Transient myeloproliferative disorder is a hematologic abnormality characterized by an uncontrolled proliferation of myeloblasts in peripheral blood and bone marrow that primarily affects newborns and babies with Down syndrome. Tumor lysis syndrome is rarely associated with transient myeloproliferative disorder.

Case presentation

Transient myeloproliferative disorder was diagnosed in a seven-day-old baby girl with Down syndrome, who was referred to our department due to hyperleukocytosis. Our patient developed tumor lysis syndrome, successfully treated with rasburicase, as a complication of transient myeloproliferative disorder resulting from rapid degradation of myeloid blasts after initiation of effective chemotherapy.

Conclusions

Tumor lysis syndrome is rarely reported as a complication of transient myeloproliferative disorder. To the best of our knowledge, this is the first case of a newborn with Down syndrome and transient myeloproliferative disorder treated with rasburicase for developing tumor lysis syndrome.

     

Delayed effect of prenatal exposure to hypoxia on the susceptibility of rats to electric seizures

We studied the delayed effects of prenatal exposure to hypoxia on the susceptibility of rats to seizures. The later was estimated using graded electroshock. The experiments were performed in two groups of 1.5-year-old male Wistar rats. The experimental group consisted of the animals that were exposed to hypoxia on day 14 of prenatal development, and the control group consisted of the animals that developed under the normal conditions. In the rats subjected to prenatal hypoxia, seizure episodes induced by weak currents in the range of 10–40 mA and their average duration were more pronounced as compared to the control animals.     

DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA

The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.