Low Concentrations of Taxol Cause Mitotic Delay Followed by Premature Dissociation of p55CDC from Mad2 and BubR1 and Abrogation of the Spindle checkpoint,Leading to Aneuploidy

Taxol is widely used for the treatment of human cancer. Its mechanism ofaction in cells is dependent on drug concentration. At low concentrations ofTaxol (5-10 nM), cells exhibit aberrant mitosis, including aneuploidy, in theabsence of mitotic arrest. At higher concentarions of Taxol (>20 nM), the cellcycle is blocked at metaphase by spindle checkpoint activation. Here wedemonstrate that low concentrations of Taxol cause mitotic delay, and result inan aneuploid population of cells after exit from mitosis. Low concentrations ofTaxol dissociated p55CDC-Mad2 or p55CDC-BubR1 complexes after mitosis,whereas high concentrations of Taxol sustained the protein complex formationleading to mitotic block. The induction of apoptosis and aneuploidy by lowconcentrations of Taxol may result from chromosome missegregation caused byspindle checkpoint defects.     

Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

Backround

Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.

Methodology/Principal Findings

Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.

Conclusion

It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.     

Dual role of topoisomerase II in centromere resolution and aurora B activity

Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.     

Repetitive Transcranial Magnetic Stimulation Attenuates the Perception of Force Output Production in Non-Exercised Hand Muscles after Unilateral Exercise

We examined whether unilateral exercise creates perception bias in the non-exercised limb and ascertained whether rTMS applied to the primary motor cortex (M1) interferes with this perception. All participants completed 4 interventions: 1) 15-min learning period of intermittent isometric contractions at 35% MVC with the trained hand (EX), 2) 15-min learning period of intermittent isometric contractions at 35% MVC with the trained hand whilst receiving rTMS over the contralateral M1 (rTMS+EX); 3) 15-min of rTMS over the ‘trained’ M1 (rTMS) and 4) 15-min rest (Rest). Pre and post-interventions, the error of force output production, the perception of effort (RPE), motor evoked potentials (MEPs) and compound muscle action potentials (CMAPs) were measured in both hands. EX did not alter the error of force output production in the trained hand (Δ3%; P>0.05); however, the error of force output production was reduced in the untrained hand (Δ12%; P<0.05). rTMS+EX and rTMS alone did not show an attenuation in the error of force output production in either hand. EX increased RPE in the trained hand (9.1±0.5 vs. 11.3±0.7; P<0.01) but not the untrained hand (8.8±0.6 vs. 9.2±0.6; P>0.05). RPE was significantly higher after rTMS+EX in the trained hand (9.2±0.5 vs. 10.7±0.7; P<0.01) but ratings were unchanged in the untrained hand (8.5±0.6 vs. 9.2±0.5; P>0.05). The novel finding was that exercise alone reduced the error in force output production by over a third in the untrained hand. Further, when exercise was combined with rTMS the transfer of force perception was attenuated. These data suggest that the contralateral M1 of the trained hand might, in part, play an essential role for the transfer of force perception to the untrained hand.     

Thermal Inactivation of Desiccation-Adapted Salmonella spp. in Aged Chicken Litter

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter was investigated in comparison with that in a nonadapted control to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85, and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction in the number of the desiccation-adapted cells in aged chicken litter with a 20% moisture content required >6, >6, ∼4 to 5, and ∼3 to 4 h of exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction in the number of nonadapted control cells in the same chicken litter was achieved within ∼1.5 to 2, ∼1 to 1.5, ∼0.5 to 1, and <0.5 h at 70, 75, 80, and 85°C, respectively. The exposure time required to obtain a 5-log reduction in the number of desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella cells survived for 50 min in chicken litter with a 20% moisture content, whereas control cells were detectable by enrichment for only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination.     

Nanoparticle Incorporation of Melittin Reduces Sperm and Vaginal Epithelium Cytotoxicity

Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001). However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42) or vaginal epithelium (p = 0.48) at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001) and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001). Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.     

Preservation of Differentiation and Clonogenic Potential of Human Hematopoietic Stem and Progenitor Cells during Lyophilization and Ambient Storage

Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25°C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.     

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1–7) and by quinuclidine motif (8), transformed into ammonium salts (9–13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC₅₀s = 0.09–1.06 µM). Transformation of 8 into salts 9–13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC₅₀∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC₅₀∼2 µM to IC₅₀∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8–13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.     

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

Background

Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.

Methods

The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.

Results and conclusion

In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.     

Probing the precision of the mitotic clock with a live-cell fluorescent biosensor

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.     

P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends.     

Hyperglycaemia Enhances Nitric Oxide Production in Diabetes: A Study from South Indian Patients

Background

We have previously reported that increased glucose levels were associated with higher serum nitric oxide (NO) levels in fructose-fed insulin resistant rats. However, the relationship between hyperglycemia and serum NO level was not clear. Therefore, the present study was designed to find the association between hyperglycemia and serum NO levels in Type 2 diabetic (T2DM) patients and T2DM with cardiovascular complication.

Methods

Endothelial cells (HUVEC) were treated with of D-glucose (10-100mM), and NO levels and NOS gene expression was measured. Hyperglycaemia was induced in Sprague-Dawley rats, and serum NO levels were measured after 8 weeks. For clinical evaluation, five groups of patients were recruited: Control (CT, n=48), Type 2 diabetes (T2DM, n=26), T2DM with hypertension (DMHT, n=46), Coronary artery diseases (CAD, n=29) and T2DM with coronary artery diseases (DMCD, n=38). NO (nitrite + nitrate) levels were measured from human serum.

Results

We found a significant (p<0.05) and dose-dependent increase in NO levels in HUVEC cells after 4 hours of high glucose exposure. eNOS and iNOS gene expression was increased in HUVEC cells after different concentrations and time periods of glucose treatment. We also observed significant (149.1±25μM, p<0.01) increase in serum NO levels in hyperglycaemic rats compared to control (76.6±13.2μM). Serum NO level was significantly higher in T2DM (111.8 μM (81.7-122.4), p<0.001) and DMCD patients ((129.4 μM (121.2-143.5), p <0.001) but not in CAD patients (76.4 μM (70.5-87)), as compared to control (68.2 μM (56.4-82.3)). We found significantly lower NO levels (83.5 μM (60.5-122.9)) in subjects suffering from diabetes since more than 5 years, compared to subjects (115.3 μM (75.2-127.1), p<0.001) with less than 5 years.

Conclusion

In conclusion, high NO levels were observed in South Indian diabetic patients. Higher glucose levels in serum might be responsible for activation of endothelial cells to enhance NO levels.     

Optimization and Validation of FePro Cell Labeling Method

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.     

Pleiotropic effects on proliferation and mineralization of primary human adipose tissue-derived stromal cells induced by simvastatin

The circulating low-density lipoprotein concentration in blood can be reduced by the administration of statins. Frequently simvastatin (SV) is prescribed. Due to the reported pleiotropic effects of SV the aim of this study was to evaluate mineralization effects on human adipose tissue-derived stromal cells upon administration of SV. After informed consent human adipose tissue-derived stromal cells were obtained from tissue surplus of regular treatments of 14 individuals. According to established protocols after adding various SV concentrations (0.01 µM, 0.1 µM, 1.0 µM, 2.0 µM), alkaline phosphate (osteoblastic marker), mineralization capability and viability were determined at day 18, 21 and 28. The Kruskal–Wallis test was performed for statistical analysis. After adding SV a dose-dependent significant decreased viability and levels of alkaline phosphatase (p < 0.01) and a significantly increased mineralization (p < 0.01) of the primary cultures was recognized during the late mineralization stage. Mineralization of the human adipose tissue-derived stromal cells was induced by SV, possibly originated from alternative pathways than the alkaline phosphatase pathway. Further investigations should be performed regarding switching into the osteoblastic differentiation and as a possible source of cells that can be used as the basis for a potential bone graft substitute, which may allow an extension of the field of application.     

Fabrication of Smart Chemical Sensors Based on Transition-Doped-Semiconductor Nanostructure Materials with μ-Chips

Transition metal doped semiconductor nanostructure materials (Sb₂O₃ doped ZnO microflowers, MFs) are deposited onto tiny µ-chip (surface area, ∼0.02217 cm²) to fabricate a smart chemical sensor for toxic ethanol in phosphate buffer solution (0.1 M PBS). The fabricated chemi-sensor is also exhibited higher sensitivity, large-dynamic concentration ranges, long-term stability, and improved electrochemical performances towards ethanol. The calibration plot is linear (r² = 0.9989) over the large ethanol concentration ranges (0.17 mM to 0.85 M). The sensitivity and detection limit is ∼5.845 µAcm⁻²mM⁻¹ and ∼0.11±0.02 mM (signal-to-noise ratio, at a SNR of 3) respectively. Here, doped MFs are prepared by a wet-chemical process using reducing agents in alkaline medium, which characterized by UV/vis., FT-IR, Raman, X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), and field-emission scanning electron microscopy (FE-SEM) etc. The fabricated ethanol chemical sensor using Sb₂O₃-ZnO MFs is simple, reliable, low-sample volume (<70.0 µL), easy of integration, high sensitivity, and excellent stability for the fabrication of efficient I–V sensors on μ-chips.     

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N₂ grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Prostate Apoptotic Induction and NFκB Suppression by Dammarolic Acid: Mechanistic Insight into Onco-Therapeutic Action of an Aglycone Asiaticoside

Prostate cancer (PCa) is addressed as the second most common form of onco-threat worldwide and is usually considered as the major cause of mortality in men. Recent times have seen a surge in exploration of plant-derived components for alternative therapeutical interventions against different oncological malignancies. Dammarolic acid or Asiatic acid (AsA) is an aglycone asiaticoside that has been reported for its efficacy in several ailments including cancer. The current study aimed to investigate the anti-proliferative potency of AsA against human prostate cancer PC-3 cells. Purified AsA was diluted and PC-3 cells were exposed to 20, 40, and 80 µM concentration and incubated for 24 h. Post-exposure, PC-3 cells showcased a substantial loss of their viability at 20 µM (p < 0.05), moreover, this reduction in cell viability escalated proportionally with an increase in AsA at concentrations of 40 and 80 µM (p < 0.01; p < 0.001) respectively. AsA-impelled loss of cellular viability was also evident from the acridine orange-stained photomicrographs, which was also used to quantify the viable and apoptotic cells using Image J software. Additionally, quantification of ROS within PC-3 cells also exhibited an increase in DCF-DA-mediated fluorescence intensity post-exposure to AsA in a dose-dependent manner. AsA-induced apoptosis in PC-3 cells was shown to be associated with augmented activity of caspase-3 proportionally to the AsA concentrations. Thus, initially, this exploratory study explicated that AsA treatment leads to anti-proliferative effects in PC-3 cells by enhancing oxidative stress and inciting apoptosis en route to onset of nuclear fragmentation.     

Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

Low-Dose Levodopa Protects Nerve Cells from Oxidative Stress and Up-Regulates Expression of pCREB and CD39

Objective

This study aimed to investigate the influence of low-dose levodopa (L-DOPA) on neuronal cell death under oxidative stress.

Methods

PC12 cells were treated with L-DOPA at different concentrations. We detected the L-DOPA induced reactive oxygen species (ROS). Meanwhile, MTT and LDH assay were performed to determine the proliferation and growth of PC12 cells with or without ROS scavenger. In addition, after pretreatment with L-DOPA at different concentrations alone or in combination with CD39 inhibitor, PC12 cells were incubated with hydrogen peroxide (H₂O₂) and the cell viability was evaluated by MTT and LDH assay. In addition, the expression of pCREB and CD39 was detected by immunofluorescence staining and Western blot assay in both cells and rat’s brain after L-DOPA treatment.

Results

After treatment with L-DOPA for 3 days, the cell proliferation and growth were promoted when the L-DOPA concentration was <30 µM, while cell proliferation was comparable to that in control group when the L-DOPA concentration was >30 µM. Low dose L-DOPA could protect the PC12 cells from H₂O₂ induced oxidative stress, which was compromised by CD39 inhibitor. In addition, the expression of CD39 and pCREB increased in both PC12 cells and rats’ brain after L-DOPA treatment.

Conclusions

L-DOPA at different concentrations has distinct influence on proliferation and growth of PC12 cells, and low dose (<30 µM) L-DOPA protects PC12 cells against oxidative stress which might be related to the up-regulation of CD39 and pCREB expression.