Galactosidase Activity Research Articles

Improved Mesenchymal Stem Cell Proliferation, Differentiation, Epithelial Transition, and Restrained Senescence on Hierarchically Patterned Porous Honey Silk Fibroin Scaffolds.

We report a significant improvement of adipose-derived mesenchymal stem cells' (ADMSCs) biocompatibility and proliferation on hierarchically patterned porous honey-incorporated silk fibroin scaffolds fabricated using a combination of soft lithography and freeze-drying techniques. Parametric variations show enhanced surface roughness, swelling, and degradation rate with good pore interconnectivity, porosity, and mechanical strength for soft-lithographically fabricated biomimetic microdome arrays on the 2% honey silk fibroin scaffold (PHSF2) as compared to its other variants, which eventually made PHSF2 more comparable to the native environment required for stem cell adhesion and proliferation. PHSF2 also exhibits sustained honey release with remarkable antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA). Honey incorporation (biochemical cue) influences microdome structural features, that is, biophysical cues (height, width, and periodicity), which further allows ADMSCs pseudopods (filopodia) to grasp the microdomes for efficient cell-cell communication and cell-matrix interaction and regulates ADMSCs behavior by altering their cytoskeletal rearrangement and thereby increases the cellular spreading area and cell sheet formation. The synergistic effect of biochemical (honey) and biophysical (patterns) cues on ADMSCs studied by the nitro blue tetrazolium assay and DCFDA fluorescence spectroscopy reveals limited free radical generation within cells. Molecular expression studies show a decrease in p53 and p21 expressions validating ADMSCs senescence inhibition, which is further correlated with a decrease in cellular senescence-associated β galactosidase activity. We also show that an increase in CDH1 and CK19 molecular expressions along with an increase in SOX9, RUNX2, and PPARγ molecular expressions supported by PHSF2 justify the substrate's efficacy of underpinning mesenchymal to epithelial transition and multilineage trans-differentiation. This work highlights the fabrication of a naturally healing nutraceutical (honey)-embedded patterned porous stand-alone tool with the potential to be used as smart stem cells delivering regenerative healing implant.

CIGARETTE SMOKE EXTRACT INDUCES DNA DAMAGE AND ACCELERATES CELLULAR SENESCENCE IN HUMAN ENDOTHELIAL CELLS

Abstract Objective: Elevated blood pressure and tobacco smoking are causes of preventable mortality. The effects of tobacco smoking on blood pressure are complex and chronic effects of tobacco smoking are inconsistent. We reported that DNA damage was increased in smokers’ blood cells compared to non-smokers. Recently, it has been reported that DNA damage is involved in cellular senescence. In this study, we investigated whether cigarette smoke extract (CSE) exerts DNA damage and cellular senescence in endothelial cells and the physiological influence of DNA damage. Design and method: The effect of CSE extracted from tobacco smoke was examined in human umbilical vein endothelial cells (HUVEC). CSE was added to HUVEC and DNA damage formation was quantified by fluorescence immunostaining; DNA single strand breaks (SSBs) with RPA 2 and double-strand breaks (DSBs) with phosphorylated histone H2AX as indices. Cellular senescence was quantified by senescence-associated -b galactosidase activity. The mRNA expression of inflammatory cytokines was quantified by real-time RT PCR. Results: SSBs increased at 24 hours after CSE stimulation, and DSBs increased significantly at 72 hours after stimulation. Continuous stimulation with CSE for 7 days resulted in an accumulation of cytosolic DNA (percentage of cells with cytosolic DNA; control: 3.4 ± 1.1, 7 days stimulation: 11.8 ± 1.8, p < 0.01)and accelerated cellular senescence (percentage of senescent cells; control: 32.5 ± 3.4, 7 days stimulation: 61.5 ± 5.2, p < 0.01). The mRNA expression of inflammatory cytokines, such as IL-6 and IL-1a was increased and prolonged by continuous CSE stimulation. Conclusions: CSE was shown to induce DNA damage, accumulation of cytosolic DNA, cellular senescence and inflammation of vascular endothelial cells.

Osteogenic capacity of mesenchymal stem cells from patients with osteoporotic hip fractures in vivo

ABSTRACT Purpose Human mesenchymal stem cells (MSCs) have the ability to differentiate into bone-forming osteoblasts. The aim of this study was to elucidate if MSCs from patients with OP show a senescent phenotype and explore their bone-forming ability in vivo. Materials and methods MSCs from patients with OP and controls with osteoarthritis (OA) were implanted into the subcutaneous tissue of immunodeficient mice for histological analysis and expression of human genes by RT-PCR. The expression of senescence-associated phenotype (SASP) genes, as well as p16, p21, and galactosidase, was studied in cultures of MSCs. Results In vivo bone formation was evaluated in 103 implants (47 OP, 56 OA). New bone was observed in 45% of the implants with OP cells and 46% of those with OA cells (p = 0.99). The expression of several bone-related genes (collagen, osteocalcin, alkaline phosphatase, sialoprotein) was also similar in both groups. There were no differences between groups in SASP gene expression, p16, and p21 expression, or in senescence-associated galactosidase activity. Conclusion Senescence markers and the osteogenic capacity in vivo of MSCs from patients with OP are not inferior to that of cells from controls of similar age with OA. This supports the interest of future studies to evaluate the potential use of autologous MSCs from OP patients in bone regeneration procedures.

Long non-coding RNA Meg3 deficiency impairs glucose homeostasis and insulin signaling by inducing cellular senescence of hepatic endothelium in obesity

Obesity-induced insulin resistance is a risk factor for diabetes and cardiovascular disease. However, the mechanisms underlying endothelial senescence in obesity, and how it impacts obesity-induced insulin resistance remain incompletely understood. In this study, transcriptome analysis revealed that the long non-coding RNA (lncRNA) Maternally expressed gene 3 (Meg3) is one of the top differentially expressed lncRNAs in the vascular endothelium in diet-induced obese mice. Meg3 knockdown induces cellular senescence of endothelial cells characterized by increased senescence-associated β–galactosidase activity, increased levels of endogenous superoxide, impaired mitochondrial structure and function, and impaired autophagy. Moreover, Meg3 knockdown causes cellular senescence of hepatic endothelium in diet-induced obese mice. Furthermore, Meg3 expression is elevated in human nonalcoholic fatty livers and nonalcoholic steatohepatitis livers, which positively correlates with the expression of CDKN2A encoding p16, an important hallmark of cellular senescence. Meg3 knockdown potentiates obesity-induced insulin resistance and impairs glucose homeostasis. Insulin signaling is reduced by Meg3 knockdown in the liver and, to a lesser extent, in the skeletal muscle, but not in the visceral fat of obese mice. We found that the attenuation of cellular senescence of hepatic endothelium by ablating p53 expression in vascular endothelium can restore impaired glucose homeostasis and insulin signaling in obesity. In conclusion, our data demonstrate that cellular senescence of hepatic endothelium promotes obesity-induced insulin resistance, which is tightly regulated by the expression of Meg3. Our results suggest that manipulation of Meg3 expression may represent a novel approach to managing obesity-associated hepatic endothelial senescence and insulin resistance.

Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical upbeta -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-upbeta -galactosidase activity using the fluorogenic substrate, C{_{12}}FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in upbeta -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p le 0.001 for the fluorogenic C{_{12}}FDG method, and r = 0.72, p le 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Apigenin Alleviates Oxidative Stress-Induced Cellular Senescence via Modulation of the SIRT1-NAD+-CD38 Axis

Oxidative stress-induced cellular senescence is now regarded as an important driving mechanism in chronic lung diseases-particularly chronic obstructive pulmonary disease (COPD). 4',5,7-trihydroxyflavone (Apigenin) is a natural flavonoid product abundantly present in fruits, vegetables, and Chinese medicinal herbs. It has been known that apigenin has anti-oxidant, anti-inflammatory and liver-protecting effects. The efficacy of apigenin for lung aging, however, has not been reported. In this study, we selected the hydrogen peroxide (H2O[Formula: see text]- or doxorubicin (DOXO)-induced senescence model in WI-38 human embryonic lung fibroblast cells to determine the potential anti-aging effects of apigenin in vitro and associated molecular mechanisms. We found that apigenin reduced senescence-associated [Formula: see text]-galactosidase (SA-[Formula: see text]-gal) activity and promoted cell growth, concomitant with a decrease in levels of Acetyl (ac)-p53, p21[Formula: see text], and p16[Formula: see text] and an increase in phospho (p)-Rb. Apigenin also increased the activation ratio of silent information regulator 1 (SIRT1), nicotinamide adenine dinucleotide (NAD[Formula: see text], and NAD[Formula: see text]/NADH and inhibited cluster of differentiation 38 (CD38) activity in a concentration-dependent manner. SIRT1 inhibition by SIRT1 siRNA abolished the anti-aging effect of apigenin. In addition, CD38 inhibition by CD38 siRNA or apigenin increased the SIRT1 level and reduced H2O2-induced senescence. Our findings suggest that apigenin is a promising phytochemical for reducing the impact of senescent cells in age-related lung diseases such as COPD.

Prevention of Fine Dust-Induced Vascular Senescence by Humulus lupulus Extract and Its Major Bioactive Compounds.

Both short- and long-term exposure to fine dust (FD) from air pollution has been linked to various cardiovascular diseases (CVDs). Endothelial cell (EC) senescence is an important risk factor for CVDs, and recent evidence suggests that FD-induced premature EC senescence increases oxidative stress levels. Hop plant (Humulus lupulus) is a very rich source of polyphenols known to have nutritional and therapeutic properties, including antioxidant behavior. The aims of this study were to evaluate whether Humulus lupulus extract prevents FD-induced vascular senescence and dysfunction and, if so, to characterize the underlying mechanisms and active components. Porcine coronary arteries and endothelial cells were treated with FD in the presence or absence of hop extract (HOP), and the senescence-associated-beta galactosidase (SA-β-gal) activity, cell-cycle progression, expression of senescence markers, oxidative stress level, and vascular function were evaluated. Results indicated that HOP inhibited FD-induced SA-β-gal activity, cell-cycle arrest, and oxidative stress, suggesting that HOP prevents premature induction of senescence by FD. HOP also ameliorated FD-induced vascular dysfunction. Additionally, xanthohumol (XN) and isoxanthohumol (IX) were found to produce the protective effects of HOP. Treatment with HOP and its primary active components XN and IX downregulated the expression of p22phox, p53, and angiotensin type 1 receptor, which all are known FD-induced redox-sensitive EC senescence inducers. Taken together, HOP and its active components protect against FD-induced endothelial senescence most likely via antioxidant activity and may be a potential therapeutic agent for preventing and/or treating air-pollution-associated CVDs.

The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence

Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor β1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated β galactosidase activity (SA-β-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-β-gal high, p=0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-β-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Aging-Affected MSC Functions and Severity of Periodontal Tissue Destruction in a Ligature-Induced Mouse Periodontitis Model.

Mesenchymal stem cells (MSCs) are known to play important roles in the repair of lost or damaged tissues and immunotolerance. On the other hand, aging is known to impair MSC function. However, little is currently known about how aged MSCs affect the host response to the local inflammatory condition and tissue deterioration in periodontitis, which is a progressive destructive disease of the periodontal tissue potentially leading to multiple tooth loss. In this study, we examined the relationship between aging-induced impairment of MSC function and the severity of periodontal tissue destruction associated with the decrease in host immunomodulatory response using a ligature-induced periodontitis model in young and aged mice. The results of micro computerized tomography (micro-CT) and histological analysis revealed a more severe bone loss associated with increased osteoclast activity in aged (50-week-old) mice compared to young (5-week-old) mice. Immunostaining analysis revealed that, in aged mice, the accumulation of inflammatory T and B cells was higher, whereas the percentage of platelet-derived growth factor receptor α (PDGFRα)+ MSCs, which are known to modulate the apoptosis of T cells, was significantly lower than in young mice. In vitro analysis of MSC function showed that the expression of surface antigen markers for MSCs (Sca-1, CD90, CD146), colony formation, migration, and osteogenic differentiation of aged MSCs were significantly declined compared to those of young MSCs. Moreover, a significantly higher proportion of aged MSCs were positive for the senescence-associated β galactosidase activity. Importantly, aged MSCs presented a decreased expression of FAS-L, which was associated with a lower immunomodulatory property of aged MSCs to induce T cell apoptosis in co-cultures compared with young MSCs. In summary, this is the first study showing that aging-induced impairment of MSC function, including immunomodulatory response, is potentially correlated with progressive periodontal tissue deterioration.

Characterization and Therapeutic Potential of Bacteriophage-Encoded Polysaccharide Depolymerases with β Galactosidase Activity against Klebsiella pneumoniae K57 Capsular Type.

Bacteriophages and phage enzymes are considered as possible alternatives to antibiotics in the treatment of infections caused by antibiotic-resistant bacteria. Due to the ability to cleave the capsular polysaccharides (CPS), one of the main virulence factors of Klebsiella pneumoniae, phage depolymerases, has potential in the treatment of K. pneumoniae infections. Here, we characterized in vivo two novel phage-encoded polysaccharide depolymerases as therapeutics against clinical isolates of K. pneumoniae. The depolymerases Dep_kpv79 and Dep_kpv767 encoded by Klebsiella phages KpV79 (Myoviridae; Jedunavirus) and KpV767 (Autographiviridae, Studiervirinae, Przondovirus), respectively, were identified as specific β-galactosidases that cleave the K. pneumoniae K57 type CPS by the hydrolytic mechanism. They were found to be highly effective at combating sepsis and hip infection caused by K. pneumoniae in lethal mouse models. Here, 80–100% of animals were protected against death by a single dose (e.g., 50 μg/mouse) of the enzyme injected 0.5 h after infection by K. pneumoniae strains of the K57 capsular type. The therapeutic effect of the depolymerases is because they strip the capsule and expose the underlying bacterium to the immune attack such as complement-mediated killing. These data provide one more confirmation that phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

The effects of pumpkin rootstock on photosynthesis, fruit mass, and sucrose content of different ploidy watermelon (Citrullus lanatus)

Pumpkin rootstock affects watermelon scion growth, fruit yield, and quality, but the mechanisms of related key enzymes and photosynthesis remain unclear. In this study, net photosynthetic rate (PN), chlorophyll (Chl) fluorescence parameters, fruit mass and sugar accumulation, and related key enzyme activities were determined during the fruit development stage in diploid and triploid watermelon lines and corresponding pumpkin rootstock-grafted lines. The results showed that pumpkin rootstock increased PN and Chl fluorescence parameters of diploid and triploid watermelon, indicating that pumpkin rootstock could increase photosynthesis, the utilization efficiency of light energy of diploid and triploid watermelon lines. Pumpkin-grafted lines had higher alkaline α-galactosidase activity and lower activities of insoluble acid invertase, sucrose phosphate synthase, and sucrose synthase than corresponding own-root lines in diploid and triploid watermelon. It indicates that pumpkin rootstock could increase the unloading of photoassimilates and reduce the conversion of photoassimilates into sucrose in diploid and triploid watermelon fruits.

Cellular and molecular mechanisms of xenobiotics-induced premature senescence.

Premature senescence, which share common features with replicative senescence such as morphology, senescence-associated galactosidase (SA-β-gal) activity, cell cycle regulation, and gene expression, can be triggered by the exposure of various xenobiotics including environmental pollutant, peroxides, and anticancer drugs. The exact mechanisms underlying the senescence onset and stabilization are still obscure. In this review, we summarized the possible cellular and molecular mechanisms of xenobiotics-induced premature senescence, including induction of reactive oxygen species (ROS), tumor suppressors, and DNA damage; disequilibrium of calcium homeostasis; activation of transforming growth factor-β (TGF-β); and blockage of aryl hydrocarbon receptor (AHR) pathway. The deeper understanding of the molecular mechanisms underlying xenobiotics-induced senescence may shed light on new therapeutic strategies for age-related pathologies and extend healthy lifespan.

Novel resveratrol derivatives have diverse effects on the survival, proliferation and senescence of primary human fibroblasts.

Resveratrol alters the cytokinetics of mammalian cell populations in a dose dependent manner. Concentrations above 25-50µM typically trigger growth arrest, senescence and/or apoptosis in multiple different cell types. In contrast, concentrations below 10µM enhance the growth of log phase cell cultures and can rescue senescence in multiple strains of human fibroblasts. To better understand the structural features that regulate these effects, a panel of 24 structurally-related resveralogues were synthesised and evaluated for their capacity to activate SIRT1, as determined by an ex-vivo SIRT1 assay, their toxicity, as measured by lactate dehydrogenase release, and their effects on replicative senescence in MRC5 human fibroblasts as measured by their effects on Ki67 immunoreactivity and senescence-associated β galactosidase activity.Minor modifications to the parent stilbene, resveratrol, significantly alter the biological activities of the molecules. Replacement of the 3,5-dihydroxy substituents with 3,5-dimethoxy groups significantly enhances SIRT1 activity, and reduces toxicity. Minimising other strong conjugative effects also reduces toxicity, but negatively impacts SIRT1 activation.At 100µM many of the compounds, including resveratrol, induce senescence in primary MRC5 cells in culture. Modifications that reduce or remove this effect match those that reduce toxicity leading to a correlation between reduction in labelling index and increase in LDH release.At 10µM, the majority of our compounds significantly enhance the growth fraction of log phase cultures of MRC5 cells, consistent with the rescue of a subpopulation of cells within the culture from senescence. SIRT1 activation is not required for rescue to occur but enhances the size of the effect.

A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

Leaching of Heavy Metals and Enzymatic Activities in Un-inoculated and Inoculated Soils with Yeast Strains

ABSTRACT Innovative biological processes that utilize the power of microorganisms are interesting tools for the treatment of Heavy metals polluted soils. This study conducted in column systems containing three different soils artificially contaminated with Cr, Zn and Cu, investigates the effect of soil bioaugmentation by two yeast strains Wickerhamomyces anomalus and Candida tropicalis on heavy metal leaching and the behavior of soil enzymatic activities. The soils have different capacity to retain heavy metals and the correlation tests showed the influence of soil properties, mainly CaCO3, clay, silt and aromaticity of organic matter on the mobilization of these heavy metals. Strong positive correlation was also observed between enzymes activities (phosphatase, galactosidase and arylsulfatase) and organic matter. The introduction of yeast strains was beneficial. It favored the immobilization of Cr and the mobilization of Cu and Zn in soils. It also stimulated the soil phosphatase, galactosidase and arylamidase activities and affected negatively arylsulfatase activity. Data thus indicate that the bioaugmetation of soils with yeast cells present an excellent performance in bioremediation by decreasing/increasing the bioavailability of metals and on soil ecosystem functioning by increasing soil enzyme activity.

Assessment of exogenous application of plant growth regulators on Cress seed germination and ?-Galactosidase activity

Plant growth regulators (PGRs) were involved in several types of abiotic stress responses by means of improving seed germination and modifying the growth and development of medicinally important Lepidium sativum via alleviating the negative effects of abiotic stresses. Therefore, the present research was carried out to investigate the effects of exogenous application of PGRson seed germination, protein content and ?-galactosidase activity of L. sativum. Germination of L. sativum seeds was monitored for a short interval after the start of incubation until growth became 100%. While cytokinin treatment showed a positive effect on seed germination more than Gibberellic acid (GA), salicylic acid (SA) produced a higher negative effect than auxins. Quantifying changes in total protein content during seed germination as influenced by PGRs revealed that all PGRs have to exert a positive effect arranged in the following order: SA ? auxin ? cytokinin ? GA. Parallel to changes in germination percentage and total protein content of seed, a negative effect was attainedon ?-galactosidase specific activity in response to PGRs with the following arrangement: SA ? auxin ? cytokinin ? GA.In conclusion, the present study proposed the potential importance of the type and magnitude of exogenously applied PGRs during the germination of easily or even more difficult-to-germinate seeds.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated β galactosidase activity. The number of β galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

Altered Glycosidase Activities at Physiological pH in the Pathogenesis of Sepsis

Glycosidases are hydrolytic enzymes that are primarily studied in the context of intracellular catabolic pathways within the lysosome. Reductions in circulating glycosidase activities have been linked to lysosomal storage diseases and are typically detected in the blood acidified to mimic lysosomal pH. There are also instances of lysosomal storage diseases linked to increased glycosidase activities in blood circulation wherein the mannose‐6‐phosphate‐dependent trafficking is rendered dysfunctional. In addition, changes in circulating glycosidase activities have been associated with other syndromes including cancer, arthritis, alcohol abuse, sepsis, and colitis. We recently discovered that glycosidases present in multiple cell types and the sera and plasma are involved in the aging of secreted and cell surface glycoproteins. The exo‐glycosidase activities of endogenous circulating glycosidases generate the stepwise loss of glycan linkages over time as glycoproteins age in circulation, sequentially exposing underlying glycan linkages starting with the removal of the terminally‐positioned sialic acids. More is known of the neuraminidases in this first step and the role of asialoglycoprotein lectin receptors that can bind and endocytose the previously underlying and cryptic galactose ligands. We have optimized the detection of glycosidases involving those with galactosidase, glucosaminidase, mannosidase, and fucosidase activities using fluorimetric substrates in blood serum and plasma at physiological pH 7.4. We found all four glycosidase activities significantly above background in the blood and plasma at normal basal levels among healthy mouse and human species. We also identify the source of these glycosidases using glycosidase‐deficient mouse strains. We further present these measurements of normality and origin in comparison with measurements made during the onset and progression of sepsis caused by different pathogens in mice and humans. Our findings to be presented include the determinations of specific activities of glycosidases in response to experimental sepsis in the mouse caused by each of the five different clinically‐derived bacterial pathogens, and the discovery of a specific change in glycosidase activity statistically linked to a poor outcome (death) in human sepsis patients.Support or Funding InformationNIH grant HL131474.NIH grant HL125352.

β-galactosidase production by Saccharomyces fragilis IZ 275 in cheese whey

Cheese whey is a byproduct of dairy industry rich in lactose, minerals and proteins which can be used as fermentation medium for microorganism. The parameters of fermentation (pH, temperature and inoculum) have not so far been optimized. Thus, the objective of this study was to investigate the temperature, pH and inoculum concentrations parameters using a Central Composite Rotational Design (CCRD) 23 and Response Surface Methodology (RSM) to ?-galactosidase production in cheese whey by Saccharomyces fragilis IZ275. The ?-galactosidase production by S. fragilis IZ 275 was evaluated through the enzymatic activity (U mL-1) using o-nitrophenyl-?-D-galactopyranoside substrate (ONPG). All the studied variables were significant and therefore the quadratic model was adequate to explain the effects of the three variables (X1, X2 and X3) on the response function (Y1 = ?-galactosidase activity). The incubation at 35 °C of temperature, pH 6.0 and 20% inoculum concentration guarantees the maximum production ?-galactosidase by Saccharomyces fragilis IZ 275 in supplemented cheese whey.

The gut microbiota metabolite urolithin A, but not other relevant urolithins, induces p53-dependent cellular senescence in human colon cancer cells

The promotion of senescence in cancer cells by dietary (poly)phenols gained attention as a promising chemopreventive strategy against colorectal (CRC) and other cancers. Urolithins (Uros) are ellagitannins and ellagic acid-derived gut microbiota metabolites that reach high concentrations in the human colon. They were postulated to be as potential anticancer agents in different CRC models, but their role as promoters of cellular senescence has never been comprehensively evaluated.We evaluated long-term senescent-mediated chemoprevention of physiologically relevant doses of different Uros and representative mixtures of human urolithin metabotypes in human CRC (HCT-116, Caco-2, and HT-29) and non-tumorigenic (CCD18-Co) cell lines. Our results show that Uro-A (but not Uro-C, IsoUro-A, or Uro-B) leads to a dose-dependent anti-clonogenic effect through the increase of the senescence-associated β–galactosidase activity, rather than by reversible cell cycle arrest and(or) apoptosis which require much higher concentrations. Senescence was accompanied by an elevated p53 and p21Cip1/Waf1 expression in HCT-116 cells (p53-wild type), but not in other CRC lines with p53 mutated or non-tumorigenic cells, which suggests that long-term senescence-mediated chemoprevention is a p53-dependent manner. Moreover, the ATP-binding cassette transporters and the phase-II metabolism of Uros limited the induction of senescence, which anticipates lower effects of conjugated Uros against systemic cancers.