The Molecular Basis of Leukocyte Adhesion Involving Phosphatidic Acid and Phospholipase D

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t_½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t_½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC₅₀ = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t_½ = 3 min at 300 nm (compared with controls of no PA, t_½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation.     

Glucose Deprivation Converts Poly(ADP-ribose) Polymerase-1 Hyperactivation into a Transient Energy-producing Process

Massive poly(ADP-ribose) formation by poly(ADP-ribose) polymerase-1 (PARP-1) triggers NAD depletion and cell death. These events have been invariantly related to cellular energy failure due to ATP shortage. The latter occurs because of both ATP consumption for NAD resynthesis and impairment of mitochondrial ATP formation caused by an increase of the AMP/ADP ratio. ATP depletion is therefore thought to be an inevitable consequence of NAD loss and a hallmark of PARP-1 activation. Here, we challenge this scenario by showing that PARP-1 hyperactivation in cells cultured in the absence of glucose (Glu⁻ cells) is followed by NAD depletion and an unexpected PARP-1 activity-dependent ATP increase. We found increased ADP content in resting Glu⁻ cells, a condition that counteracts the increase of the AMP/ADP ratio during hyperpoly(ADP-ribosyl)ation and preserves mitochondrial coupling. We also show that the increase of ATP in Glu⁻ cells is due to adenylate kinase activity, transforming AMP into ADP which, in turn, is converted into ATP by coupled mitochondria. Interestingly, PARP-1-dependent mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome complex (Cyt c) is reduced in Glu⁻ cells, even though cell death eventually occurs. Overall, the present study identifies basal ADP content and adenylate kinase as key determinants of bioenergetics during PARP-1 hyperactivation and unequivocally demonstrates that ATP loss is not metabolically related to NAD depletion.     

Changes from High Potassium (HK) to Low Potassium (LK) in Bovine Red Cells

Red cells of newborn calves contain 105–110 mmole K⁺ and 1–5 mmole Na⁺ per liter of cells. As the animals age the K⁺ content decreases to a value of 25–30 mmole/liter of cells after about 60 days. At approximately the same time, the sodium content reaches a value of 60–70 mmole/liter. The time required for half change (t_½) is 35–37 days for both Na⁺ and K⁺. The activity of (Na + K)-adenosine triphosphatase (ATPase) and the influx of K⁴² and Rb⁸⁶ into the red cells are high at birth and are reduced to 5 and 15% of their original values, respectively, in mature animals. t_½ for both is of the order of 30–35 days. The membrane Mg-ATPase activity is also high at birth and is reduced with a t_½ of 28–32 days to a final value of about 20% of its activity at birth. Separation of red cells according to their age showed that, in animals at the age of transition, newly formed red cells contain a higher K/Na ratio and a higher active transport capacity than older red cells of the same animal. It is suggested that the changes observed are a reflection of the average age of the red cell population as the animal grows.     

The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain

The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa₃ and c₅₄₇ have unaltered oxidation half-times, designated t_{½ on}, as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t_{½ off}, decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t_{½ on}, for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t_{½ off} decreases only 30% while the extent of oxidation decreases 50%. The values of t_{½ on} and t_{½ off} and the extent of oxidation of cytochrome b₅₅₃ and flavoprotein Fp_ha were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:     

SCFFbxw15 Mediates Histone Acetyltransferase Binding to Origin Recognition Complex (HBO1) Ubiquitin-Proteasomal Degradation to Regulate Cell Proliferation

Histone acetyltransferase binding to origin recognition complex (HBO1) plays a crucial role in DNA replication licensing and cell proliferation, yet its molecular regulation in cells is relatively unknown. Here an uncharacterized protein, Fbxw15, directly interacts with HBO1, a labile protein (t_½ = ∼3 h), to mediate its ubiquitination (Lys³³⁸) and degradation in the cytoplasm. Fbxw15-mediated HBO1 depletion required mitogen-activated protein kinase 1 (Mek1), which was sufficient to trigger HBO1 phosphorylation and degradation in cells. Mek1 ability to produce HBO1 degradation was blocked by Fbxw15 silencing. Lipopolysaccharide induced HBO1 degradation, an effect abrogated by Fbxw15 or Mek1 cellular depletion. Modulation of Fbxw15 levels was able to differentially regulate histone H3K14 acetylation and cellular proliferation by altering HBO1 levels. These studies authenticate Fbxw15 as a ubiquitin E3 ligase subunit that mediates endotoxin-induced HBO1 depletion in cells, thereby controlling cell replicative capacity.     

Water transport in the midrib tissue of maize leaves : direct measurement of the propagation of changes in cell turgor across a plant tissue

Water movement across plant tissues occurs along two paths: from cell-to-cell and in the apoplasm. We examined the contribution of these two paths to the kinetics of water transport across the parenchymatous midrib tissue of the maize (Zea mays L.) leaf. Water relations parameters (hydraulic conductivity, Lp; cell elastic coefficient, ε; half-time of water exchange for individual cells, T_½) of individual parenchyma cells determined with the pressure probe varied in different regions of the midrib. In the adaxial region, Lp = (0.3 ± 0.3)·10⁻⁵ centimeters per second per bar, ε = 103 ± 72 bar, and T_½ = 7.9 ± 4.8 seconds (n = seven cells); whereas, in the abaxial region, Lp = (2.5 ± 0.9)·10⁻⁵ centimeters per second per bar, ε = 41 ± 9 bar, and T_½ = 1.3 ± 0.5 seconds (n = 7). This zonal variation in Lp, ε, and T_½ indicates that tissue inhomogeneities exist for these parameters and could have an effect on the kinetics of water transport across the tissue.

The diffusivity of the tissue to water (D_t) obtained from the sorption kinetics of rehydrating tissue was D_t = (1.1 ± 0.4)·10⁻⁶ square centimeters per second (n = 6). The diffusivity of the cell-to-cell path (D_c) calculated from pressure probe data ranged from D_c = 0.4·10⁻⁶ square centimeters per second in the adaxial region to D_c = 6.1·10⁻⁶ square centimeters per second in the abaxial region of the tissue. D_t D_c suggests substantial cell-to-cell transport of water occurred during rehydration. However, the tissue diffusivity calculated from the kinetics of pressure-propagation across the tissue (D_t′) was D_t′ = (33.1 ± 8.0)·10⁻⁶ square centimeters per second (n = 8) and more than 1 order of magnitude larger than D_t. Also, the hydraulic conductance of the midrib tissue (Lp_m per square centimeter of surface) estimated from pressure-induced flows across several parenchyma cell layers was Lp_m = (8.9 ± 5.6)·10⁻⁵ centimeters per second per bar (n = 5) and much larger than Lp.

These results indicate that the preferential path for water transport across the midrib tissue depends on the nature of the driving forces present within the tissue. Under osmotic conditions, the cell-to-cell path dominates, whereas under hydrostatic conditions water moves primarily in the apoplasm.

     

Effect of Physical Parameters on the In Situ Survival of Escherichia coli MC-6 in an Estuarine Environment

Survival of Escherichia coli MC-6 of fecal origin in an estuarine environment as affected by time, water temperature, dissolved oxygen, salinity, and montmorillonite in diffusion chambers has been elucidated. Several in situ physical parameters were recorded simultaneously, and viable cell numbers were estimated. The survival of the bacteria varied seasonally. Montmorillonite addition extended the time needed for a 50% reduction of the viable cell population (t_½) of cells by 40% over the t_½ of cells in Rhode River water alone. The effect of this clay was not significantly greater between 50- to 1,000-μg/ml montmorillonite concentrations. In all experiments, the relationships among pairs of variables were studied by regression and correlation analysis. The slope between viable cell numbers and water temperatures increased about 50% for each 10 C increment in temperature and gave a correlation coefficient r = 0.617, significant at 95% confidence level. A similar correlation coefficient, r = 0.670, was obtained between water temperature and t_½ of the initial cell population. In all experiments regressions were performed considering all variables after bacteria had been in the Rhode River environment for 3 days. Coefficient of multiple determinaton was estimated as R² = 0.756. Approximately 75.6% of the variance of viable cell numbers can be explained by variation in water temperature, dissolved oxygen, and salinity. Simple correlation coefficients within the regression steps were also computed. Survival of bacteria was closely and negatively correlated with increasing water temperature (r = -0.717). It is suggested that water temperature is the most important factor in predicting fecal coliform survival from point and nonpoint sources in assessing water quality in an estuarine ecosystem.     

Molecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4

The secreted form of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes a key reaction in intracellular NAD biosynthesis, acts as a damage-associated molecular pattern triggering Toll-like receptor 4 (TLR4)-mediated inflammatory responses. However, the precise mechanism of interaction is unclear. Using an integrated approach combining bioinformatics and functional and structural analyses, we investigated the interaction between NAMPT and TLR4 at the molecular level. Starting from previous evidence that the bacterial ortholog of NAMPT cannot elicit the inflammatory response, despite a high degree of structural conservation, two positively charged areas unique to the human enzyme (the α1-α2 and β1-β2 loops) were identified as likely candidates for TLR4 binding. However, alanine substitution of the positively charged residues within these loops did not affect either the oligomeric state or the catalytic efficiency of the enzyme. The kinetics of the binding of wildtype and mutated NAMPT to biosensor-tethered TLR4 was analyzed. We found that mutations in the α1-α2 loop strongly decreased the association rate, increasing the K_D value from 18 nM, as determined for the wildtype, to 1.3 μM. In addition, mutations in the β1-β2 loop or its deletion increased the dissociation rate, yielding K_D values of 0.63 and 0.22 μM, respectively. Mutations also impaired the ability of NAMPT to trigger the NF-κB inflammatory signaling pathway in human cultured macrophages. Finally, the involvement of the two loops in receptor binding was supported by NAMPT-TLR4 docking simulations. This study paves the way for future development of compounds that selectively target eNAMPT/TLR4 signaling in inflammatory disorders.     

NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective,PAR-independent metabolic catastrophe and cell death induced by β-lapachone

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD⁺ synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD⁺ pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD⁺ consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD⁺ synthesis while increasing NAD⁺ consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)⁺ depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD⁺-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD⁺ synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD⁺ pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.An emerging metabolic target for the treatment of recalcitrant cancers, such as pancreatic adenocarcinoma (PDA), is their reliance on NAD⁺ synthesis, particularly through the nicotinamide-recycling pathway.^{1, 2, 3} Rapid and efficient NAD⁺ synthesis is critical to sustain signaling processes, such as deacetylation by sirtuins and adenosine diphosphate (ADP) ribosylation by poly(ADP ribose) polymerase 1 (PARP1). NAD(P)⁺ pools are also necessary to support anabolic metabolism and proliferation of cancer cells. In an attempt to leverage increased tumor-cell reliance on NAD⁺ synthesis, small molecule inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) were developed (e.g., FK866).⁴ NAMPT catalyzes the rate-limiting step of the most active pathway of NAD⁺ synthesis. Inhibitors of NAMPT, such as FK866, reduce NAD⁺ levels, induce canonical apoptosis preferentially in cancer cells in vitro, inhibit tumor growth, and increase overall survival in preclinical cancer models.^{1, 5, 6, 7} FK866 (APO866) was relatively well tolerated in humans and advanced to phase II clinical trials. However, owing to its short half-life in circulation, prolonged treatment regimens were required and toxicity to normal, rapidly proliferating hematopoietic cells was noted. Accordingly, FK866 and other NAMPT inhibitors did not demonstrate sufficient tumor-selectivity to achieve clinical success as single agents.⁸To increase the specificity and efficacy of NAMPT inhibition, we combined FK866 with β-lapachone (β-lap), a targeted cancer therapeutic that causes tumor-selective PARP1 hyperactivation and NAD⁺ depletion in an NADPH:quinone oxidoreductase 1 (NQO1)-specific manner.⁹ β-Lap is a substrate for two-electron oxidoreduction by NQO1, a Phase II quinone-detoxifying enzyme.⁹ The resulting hydroquinone form of β-lap is highly unstable and spontaneously reacts with oxygen to revert back to the parent compound, generating two moles of superoxide per mole of NAD(P)H used in the process. This results in a futile cycle that occurs rapidly in NQO1-overexpressing cells, causing marked NADH/NADPH oxidation. DNA damage in the form of base oxidation and DNA single-strand breaks results from H₂O₂ generated from the futile redox cycle. In an attempt to repair this damage, PARP1 becomes hyperactivated, generating extensive branched poly(ADP ribose) (PAR) polymer. Hyperactivated PARP1 substantially depletes NAD⁺ and ultimately adenosine triphosphate (ATP) levels, thereby inhibiting subsequent repair of β-lap-induced DNA lesions. The observed cell death is caspase-independent and driven by nuclear translocation of apoptosis-inducing factor (AIF), activation of μ-calpain, and post-translational modification of GAPDH.^{10, 11, 12, 13} NQO1 is highly expressed in many types of cancer, and the therapeutic window provided by NQO1 bioactivation of β-lap has advanced its use to phase I clinical trials (ARQ761).¹⁴ Elevated NQO1 expression (≥10-fold) has been identified in ~85% of patient tissue from pancreatic ductal adenocarcinoma (PDA), making pancreatic cancer an especially appealing target for therapy using NQO1 bioactivatable drugs, such as β-lap.^{15, 16, 17, 18} However, dose-limiting methemoglobinemia caused by nonspecific reactive oxygen species (ROS) generation at high β-lap doses may limit the efficacy of β-lap as monotherapy.¹⁹ Strategies for increasing cancer cell cytotoxicity while maintaining NQO1 specificity could enhance use of β-lap for therapy against PDAs, as well as other solid cancers that overexpress NQO1.We found that examining cell death pathways induced by β-lap, with or without FK866 treatment, is a novel means to elucidate general mechanisms of lethality mediated by NAD⁺ loss, as cell death by PARP1 hyperactivation occurs in other contexts. Notably, cell death induced by ischemia/reperfusion shares many of the same characteristics: ROS induction, PARP1 hyperactivation, calcium release, AIF translocation, and caspase-independence.^{20, 21} Similarly, treatment with methylnitronitrosoguanidine (MNNG; a DNA alkylating agent) or induction of neuronal excitotoxicty induces PARP1 hyperactivation and cell death, but without futile cycle-induced ROS production.^{22, 23, 24} Recent studies suggest an important role for accumulated free PAR polymer that can directly activate μ-calpain, activate and release AIF, and inhibit glycolysis.^{22, 25, 26, 27, 28} By combining β-lap and FK866, we uncouple NAD⁺ and ATP depletion from the robust formation of PAR noted with β-lap alone, allowing us to define the function of PAR formation in β-lap-induced cell death.β-Lap and FK866 have distinct, but highly complementary mechanisms of action. β-Lap induces tumor-selective NAD⁺ depletion specifically in cancer cells that express high levels of NQO1. FK866 primes cancer cells for cell death by lowering NAD⁺/NADH pools and prevents recovery by inhibiting NAD⁺ synthesis from nicotinamide liberated by activated PARP1. We show that the increased dependence of PDA cells on glycolysis is specifically targeted by ROS-induced, NAD⁺ depletion caused by exposure to both drugs. Glycolytic inhibition, ATP depletion, and cell death is independent of PAR formation, strongly suggesting that PAR accumulation is not directly involved. The use of β-lap with NAMPT inhibitors results in synergistic NQO1- and PARP1-dependent cancer cell death, allowing the use of lower doses and shorter treatment times for both therapeutics.     

Turnover and uptake by organs of radioactive serum high-density lipoprotein cholesteryl esters and phospholipids in the rat in vivo

The serum decay of rat serum high-density lipoprotein (HD lipoprotein), labelled biosynthetically with ³²P in the phospholipid or with ³H in the cholesteryl ester moiety, was measured in rats after partial hepatectomy or sham operation. The serum decay of ³H-labelled HD lipoprotein cholesteryl esters was biexponential. In sham-operated rats the t_½ values for the rapid phase and the slow phase were 0.2±0.1h and 4.2±0.4h (means±s.e.m.) respectively. After removal of two-thirds of the liver the t_½ value of the rapid phase did not change (0.1±0.1h), whereas the t_½ value of the slow phase increased to 5.7±0.8h. Partial hepatectomy hardly changed extrahepatic tissue radioactivities, whereas the percentage of the injected dose recovered in the liver 6h after injection decreased from 34.0±1.9% before to 13.5±1.6% after partial hepatectomy. The ³²P-labelled HD lipoprotein phospholipids showed a rapid monoexponential decay from serum with t_½ values of 0.71±0.3h and 1.48±0.11h after sham operation or partial hepatectomy respectively. The tissue ³²P radioactivities in the shamoperated rats, measured 1h after injection, were 46.0±1.7% (liver), 1.7±0.3% (adipose tissue), 3.7±1.2% (skeletal muscle) and 3.0±0.0% (erythrocytes) of the injected dose. Only the value for liver was affected by partial hepatectomy and decreased to 16.7±3.8%. In a previous publication [Van Tol, Van Gent, Van't Hooft & Vlaspolder (1978) Atherosclerosis 29, 439–448] we showed in a highly comparable experimental setting that the turnover rates of HD apolipoproteins A and C in vivo are not influenced by removal of two-thirds of the liver. From the present study it is clear that the removal rates of radioactive HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids from serum in vivo are decreased by partial hepatectomy. The results indicate the possibility of partly separate metabolic pathways of HD apolipoproteins A and C, HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids. The phospholipids and cholesteryl esters of HD lipoprotein are metabolized predominantly by the liver. Possible mechanisms for the hepatic uptake and metabolism of HD lipoprotein cholesteryl (esters) and phospholipids are discussed.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD⁺) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD⁺ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.Subject terms: Drug development, Cancer metabolism

     

AMPK mediates a pro-survival autophagy downstream of PARP-1 activation in response to DNA alkylating agents

In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax?/? Bak?/? double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.     

Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain

Introduction

Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.

Methods

Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E₂ (PGE₂), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.

Results

Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE₂, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.

Conclusions

These results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.     

Photosynthetic Adaptation by Synechococcus leopoliensis in Response to Exogenous Dissolved Inorganic Carbon

Synechococcus leopoliensis was grown over a wide range of dissolved inorganic carbon (DIC) concentrations (4-25,000 micromolar) which were obtained by varying culture pH (6.2-9.6) and the CO₂ concentration of the gas stream (36-50,000 microliters per liter). The [DIC] required to half-saturate photosynthesis (K_½^DIC) was found to vary depending upon the ambient DIC concentration at which the cells were grown. Low [DIC] grown cells exhibited low values of K_½^DIC (4.7 micromolar) whereas cells grown at high [DIC] exhibited high values of K_½^DIC (1-2.5 millimolar). Intermediate concentrations of DIC produced intermediate values. Changes in K_½^DIC appeared to be solely a function of [DIC] and were independent of both culture pH and CO₂ concentration. As changes in K_½^DIC occur in response to DIC concentrations commonly found in natural systems we suggest this adaptation may be of ecological significance.     

SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin

Cadmium is one of the most toxic metal compounds found in the environment. It is well established that Cd induces hepatotoxicity in humans and multiple animal models. Melatonin, a major secretory product of the pineal gland, has been reported to protect against Cd-induced hepatotoxicity. However, the mechanism behind this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd induced mitochondrial-derived superoxide anion-dependent autophagic cell death. Specifically, Cd decreased SIRT3 protein expression and activity and promoted the acetylation of SOD2, superoxide dismutase 2, mitochondrial, thus decreasing its activity, a key enzyme involved in mitochondrial ROS production, although Cd did not disrupt the interaction between SIRT3 and SOD2. These effects were ameliorated by overexpression of SIRT3. However, a catalytic mutant of SIRT3 (SIRT3^H248Y) lacking deacetylase activity lost the capacity to suppress Cd-induced autophagy. Notably, melatonin treatment enhanced the activity but not the expression of SIRT3, decreased the acetylation of SOD2, inhibited mitochondrial-derived O₂^•− production and suppressed the autophagy induced by 10 μM Cd. Moreover, 3-(1H-1,2,3-triazol-4-yl)pyridine, a confirmed selective SIRT3 inhibitor, blocked the melatonin-mediated suppression of autophagy by inhibiting SIRT3-SOD2 signaling. Importantly, melatonin suppressed Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin exerts a hepatoprotective effect on mitochondrial-derived O₂^•−-stimulated autophagic cell death that is dependent on the SIRT3/SOD2 pathway.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Two components of auxin transport

The transport of indoleacetic acid-1-¹⁴C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t_½ of about 30 minutes) and a slow transport system (t_½ about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.