A chip-based system mimicking the transport function of the human cardiovascular system has been established at minute but standardized microsystem scale. A peristaltic on-chip micropump generates pulsatile shear stress in a widely adjustable physiological range within a microchannel circuit entirely Time-lapse and 3D imaging two-photon microscopy were used to visualise details of spatiotemporal adherence of the endothelial cells to the channel system and to each other. The first indicative long-term experiments revealed stable performance over two and four weeks. The potential application of this system for the future establishment of human-on-a-chip systems and basic human endothelial cell research is discussed.
Edited by John M. Denu NADH (NAD ؉) is an essential metabolite involved in various cellular biochemical processes. The regulation of NAD ؉ metabolism is incompletely understood. Here, using budding yeast (Saccharomyces cerevisiae), we established an NAD ؉ intermediate-specific genetic system to identify factors that regulate the de novo branch of NAD ؉ biosynthesis. We found that a mutant strain (mac1⌬) lacking Mac1, a copper-sensing transcription factor that activates copper transport genes during copper deprivation, exhibits increases in quinolinic acid (QA) production and NAD ؉ levels. Similar phenotypes were also observed in the hst1⌬ strain, deficient in the NAD ؉-dependent histone deacetylase Hst1, which inhibits de novo NAD ؉ synthesis by repressing BNA gene expression when NAD ؉ is abundant. Interestingly, the mac1⌬ and hst1⌬ mutants shared a similar NAD ؉ metabolism-related gene expression profile, and deleting either MAC1 or HST1 de-repressed the BNA genes. ChIP experiments with the BNA2 promoter indicated that Mac1 works with Hst1-containing repressor complexes to silence BNA expression. The connection of Mac1 and BNA expression suggested that copper stress affects de novo NAD ؉ synthesis, and we show that copper stress induces both BNA expression and QA production. Moreover, nicotinic acid inhibited de novo NAD ؉ synthesis through Hst1-mediated BNA repression, hindered the reuptake of extracellular QA, and thereby reduced de novo NAD ؉ synthesis. In summary, we have identified and characterized novel NAD ؉ homeostasis factors. These findings will expand our understanding of the molecular basis and regulation of NAD ؉ metabolism. NAD ϩ and its reduced form NADH are primary redox carriers in cellular metabolism. NAD ϩ is also a cosubstrate in protein modifications, such as protein deacetylation mediated by the sirtuins (Sir2 family proteins) and ADP-ribosylation mediated by the poly(ADP-ribose) polymerases. These protein modifications contribute to the maintenance and regulation of chromatin structure, DNA repair, circadian rhythm, metabolic responses, and life span (1-4). NAD ϩ is also an NADP ϩ precursor, which, like NAD ϩ , is carefully balanced with its reduced form NADPH to maintain a favorable redox state. Aberrant NAD ϩ metabolism is associated with a number of diseases, including diabetes, cancer, and neuron degeneration (2, 3, 5-11). Administration of NAD ϩ precursors, such as nicotinamide mononucleotide (NMN), 2 nicotinamide (NAM), nicotinic acid riboside, and nicotinamide riboside (NR), has been shown to ameliorate deficiencies related to aberrant NAD ϩ metabolism in yeast, mouse, and human cells (3, 5-10, 12-15). However, the molecular mechanisms underlying the beneficial effects of NAD ϩ precursor supplementation are not yet completely understood. The NAD ϩ pool is maintained by multiple NAD ϩ biosynthesis pathways, which are conserved from bacteria to humans. Depending on the cell types, growth conditions, and availability of specific NAD ϩ precursors, one pathway may dominate the others. ...
Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite with wide-ranging and significant roles in the cell. Defects in NAD+ metabolism have been associated with many human disorders; it is therefore an emerging therapeutic target. Moreover, NAD+ metabolism is perturbed during colonization by a variety of pathogens, either due to the molecular mechanisms employed by these infectious agents or by the host immune response they trigger. Three main biosynthetic pathways, including the de novo and salvage pathways, contribute to the production of NAD+ with a high degree of conservation from bacteria to humans. De novo biosynthesis, which begins with l-tryptophan in eukaryotes, is also known as the kynurenine pathway. Intermediates of this pathway have various beneficial and deleterious effects on cellular health in different contexts. For example, dysregulation of this pathway is linked to neurotoxicity and oxidative stress. Activation of the de novo pathway is also implicated in various infections and inflammatory signaling. Given the dynamic flexibility and multiple roles of NAD+ intermediates, it is important to understand the interconnections and cross-regulations of NAD+ precursors and associated signaling pathways to understand how cells regulate NAD+ homeostasis in response to various growth conditions. Although regulation of NAD+ homeostasis remains incompletely understood, studies in the genetically tractable budding yeast Saccharomyces cerevisiae may help provide some molecular basis for how NAD+ homeostasis factors contribute to the maintenance and regulation of cellular function and how they are regulated by various nutritional and stress signals. Here we present a brief overview of recent insights and discoveries made with respect to the relationship between NAD+ metabolism and selected human disorders and infections, with a particular focus on the de novo pathway. We also discuss how studies in budding yeast may help elucidate the regulation of NAD+ homeostasis.
For an adjusted platelet concentration of 50,000 platelets·μL-1, both colorimetric assays (ACP and LDH) allowed a similar accurate quantification of the mean platelet density compared to the microscopic evaluation. Better linearity of the assay standards, less variability of the results and a lower influence of platelet activation on the measurements mark the ACP assay as more suitable for the assessment of material surface adherent platelets compared to the LDH assay, particularly, if near physiological platelet concentrations are applied.
NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.
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