Transport governs flow-enhanced cell tethering through L-selectin at threshold shear

Flow-enhanced cell adhesion is a counterintuitive phenomenon that has been observed in several biological systems. Flow augments L-selectin-dependent adhesion by increasing the initial tethering of leukocytes to vascular surfaces and by strengthening their subsequent rolling interactions. Tethering or rolling might be influenced by physical factors that affect the formation or dissociation of selectin-ligand bonds. We recently demonstrated that flow enhanced rolling of L-selectin-bearing microspheres or neutrophils on P-selectin glycoprotein ligand-1 by force decreased bond dissociation. Here, we show that flow augmented tethering of these microspheres or cells to P-selectin glycoprotein ligand-1 by three transport mechanisms that increased bond formation: sliding of the sphere bottom on the surface, Brownian motion, and molecular diffusion. These results elucidate the mechanisms for flow-enhanced tethering through L-selectin.     

Cytolinker cross-talk: periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.     

Magnetic alignment in warthogs Phacochoerus africanus and wild boars Sus scrofa

Magnetic alignment (MA) results from the preference of animals to align themselves along the field lines of the geomagnetic field, a behavioural expression of a magnetic sense. MA is well documented for ruminants and might demonstrate a general magnetic sensory ability among artiodactyls. We measured body‐axis alignment in 1614 foraging or resting wild boars Sus scrofa, 1849 wild boar beds, and 1347 warthogs Phacochoerus africanus, and found a highly significant north–south preference. The magnetic field was the only common denominator of all observations. Thus, we provide the first data suggesting a magnetic sense in the Suidae.     

Statistical vs. Stochastic experimental design: An experimental comparison on the example of protein refolding

Optimization of experimental problems is a challenging task in both engineering and science. In principle, two different design of experiments (DOE) strategies exist: statistical and stochastic methods. Both aim to efficiently and precisely identify optimal solutions inside the problem‐specific search space. Here, we evaluate and compare both strategies on the same experimental problem, the optimization of the refolding conditions of the lipase from Thermomyces lanuginosus with 26 variables under study. Protein refolding is one of the main bottlenecks in the process development for recombinant proteins. Despite intensive effort, the prediction of refolding from sequence information alone is still not applicable today. Instead, suitable refolding conditions are typically derived empirically in large screening experiments. Thus, protein refolding should constitute a good performance test for DOE strategies. We compared an iterative stochastic optimization applying a genetic algorithm and a standard statistical design consisting of a D‐optimal screening step followed by an optimization via response surface methodology. Our results revealed that only the stochastic optimization was able to identify optimal refolding conditions (～1.400 U g^?1 refolded activity), which were 3.4‐fold higher than the standard. Additionally, the stochastic optimization proved quite robust, as three independent optimizations performed similar. In contrast, the statistical DOE resulted in a suboptimal solution and failed to identify comparable activities. Interactions between process variables proved to be pivotal for this optimization. Hence, the linear screening model was not able to identify the most important process variables correctly. Thereby, this study highlighted the limits of the classic two‐step statistical DOE. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Thermolabile duplex-specific nuclease

Using random mutagenesis of the gene encoding duplex-specific nuclease from the king crab we found a new mutant that retained all properties of the wild-type protein, but exhibited a much lower thermal stability. This enzyme, denoted thermolabile duplex-specific nuclease (DSN-TL), exhibits high processivity and selective cleavage of dsDNA. The inactivation temperature for DSN-TL is 15–20°C lower than that of the widely used DNase I and shrimp nuclease, and its catalytic activity is more than 10 times higher. Moreover, DSN-TL is resistant to proteinase K treatment. These properties make DSN-TL very useful for removing genomic DNA from RNA samples intended for quantitative RT-PCR.     

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD⁺ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.     

Prenatal Monosodium Glutamate Causes Long-Lasting Cholinergic and Adrenergic Changes in Various Brain Regions

Prenatal monosodium glutamate (MSG) given through the mother's diet was found previously to cause behavioral changes in the offspring, including learning disabilities. In the present study, neurochemical parameters were measured in the brains of prenatally exposed rats at various ages throughout development up to adulthood. At 15 days of age, choline uptake and choline acetyltransferase (ChAT) activity in the frontal cortex were significantly reduced (by 80 and 25%, respectively) in MSG-exposed animals, whereas the same cholinergic parameters in hippocampus were not changed. During later development, choline uptake gradually increased, until in adulthood it became significantly higher in MSG-exposed animals than in the controls. This enhancement was found in both males and females. Our previous study showed that only the male offspring were learning disabled. Choline uptake and ChAT activity were enhanced in the hippocampus of adult male animals. Norepinephrine (NE) uptake was reduced (by 25%) in the frontal cortex of males only. There was no change in NE uptake in the hypothalamus.     

Cellular responses of two Latin-American cultivars of Lotus corniculatus to low pH and Al stress

Toxic effects of acidic root medium and aluminium were evaluated in two forage cultivars of Lotus corniculatus differing in their tolerance to Al stress. The structural response of most of the root cells exposed to low pH without Al³⁺ differed markedly from that induced by the combined stress. Conspicuous alteration of the nucleus was present only at low pH 4.0 and disintegration of the cytoplasmic components was more drastic than in the roots exposed to acidic solution containing Al³⁺. Cells exposed to low pH without Al, did not produce wall thickenings. Severely damaged cytoplasm and localized death in some cortical cells or groups of cells contrasting with almost intact cells exposed to Al³⁺ stress were found. In this respect, a strong correlation between the occurrence of cell wall thickenings and a better preserved structure of the cytoplasm was observed. The frequency of cell damage in the more tolerant cultivar UFRGS was generally lower, significantly more cortical cells capable of maintaining their resting membrane potential were present than in the sensitive INIA Draco. The difference in their tolerance is related rather to the exudation of citrate and oxalate that was higher in UFRGS than to the accumulation of tannins, which increased after Al treatment in both cultivars.     

Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.