Dependence Of Enzyme Activity Research Articles

Xanthan: enzymatic degradation and novel perspectives of applications.

The extracellular heteropolysaccharide xanthan, synthesized by bacteria of the genus Xanthomonas, is widely used as a thickening and stabilizing agent across the food, cosmetic, and pharmaceutical sectors. Expanding the scope of its application, current efforts target the use of xanthan to develop innovative functional materials and products, such as edible films, eco-friendly oil surfactants, and biocompatible composites for tissue engineering. Xanthan-derived oligosaccharides are useful as nutritional supplements and plant defense elicitors. Development and processing of such new functional materials and products often necessitate tuning of xanthan properties through targeted structural modification. This task can be effectively carried out with the help of xanthan-specific enzymes. However, the complex molecular structure and intricate conformational behavior of xanthan create problems with its enzymatic hydrolysis or modification. This review summarizes and analyzes data concerning xanthan-degrading enzymes originating from microorganisms and microbial consortia, with a particular focus on the dependence of enzymatic activity on the structure and conformation of xanthan. Through a comparative study of xanthan-degrading pathways found within various bacterial classes, different microbial enzyme systems for xanthan utilization have been identified. The characterization of these new enzymes opens new perspectives for modifying xanthan structure and developing innovative xanthan-based applications. KEY POINTS: • The structure and conformation of xanthan affect enzymatic degradation. • Microorganisms use diverse multienzyme systems for xanthan degradation. • Xanthan-specific enzymes can be used to develop xanthan variants for novel applications.

CoCl2 -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.

SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD+ -dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2 -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl2 -induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.

Analysis of the activity of oxidative enzymes by multivariate regression in the presence of Mg<sup>2+</sup> oyster mushroom mycelium

Studies have been conducted to optimize the process of deep cultivation of the Oyster mushroom mycelium. The process of obtaining mycelium as a seed for cultivation of fruiting bodies of mushrooms has been improved. The effect of different concentrations of magnesium carbonate (Magnesium carbonates) on the growth characteristics of mycelium of the fungus Pleurotus ostreatus during deep cultivation has been studied. The dependence of enzyme activity on the concentration of metal in the nutrient medium of germinating mycelium of oyster mushrooms has been revealed. The method for determining the activity of catalase by spectrophotometric method has been adapted for the studied objects. For the first time the data on the activity of mycelium catalase and superoxide dismutase enzymes in the presence of magnesium carbonate additive have been obtained. It has been found that the application of magnesium carbonate in low concentrations has a positive effect on the growth of mycelial biomass of the fungus Pleurotus ostreatus, since with increasing concentration (Mg2+) a decrease in biomass growth rate and catalase activity has been observed, presumably due to the participation of magnesium in creating a certain ionic concentration at which catalase inactivation begins. The possibility of applying the method of multivariate regression in the form of the principal components analysis (PCA) has been studied. The redox state of Pleurotus ostreatus culture at the level of enzyme components of the antioxidant defense system during submerged cultivation of basidomycetes has been analyzed, which showed how the obtained variables with different measurement units are interconnected. The account graphs also clearly indicate the dependence of mycelial growth on the concentration of the additive used. The introduction of the cultivation conditions proposed in this work in the practice of mushroom production potentially contributes to a more successful resistance of macromycetes to biotic and abiotic stress. The results of the research are relevant to the development of the fundamentals of the science of fungi.

Heavy metal(loid) effect on multi-biomarker responses in apex predator: Novel assays in the monitoring of white stork nestlings

The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence–based oxidative stress biomarkers, metallothionein levels, glutathione–dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first–time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.

Albumin alleviated esketamine-induced neuronal apoptosis of rat retina through downregulation of Zn2+-dependent matrix metalloproteinase 9 during the early development

AimsEsketamine upregulates Zn2+-dependent matrix metalloproteinase 9 (MMP9) and increases the neuronal apoptosis in retinal ganglion cell layer during the early development. We aimed to test whether albumin can alleviate esketamine-induced apoptosis through downregulating Zn2+-dependent MMP9.MethodsWe investigate the role of Zn2+ in esketamine-induced neuronal apoptosis by immunofluorescence. MMP9 protein expression and enzyme activity were investigated by zymography in situ., western blot and immunofluorescence. Whole-mount retinas from P7 Sprague-Dawley rats were used.ResultsWe demonstrated that esketamine exposure increased Zn2+ in the retinal GCL during the early development. Zn2+-dependent MMP9 expression and enzyme activity up-regulated, which eventually aggravated apoptosis. Albumin effectively down-regulated MMP9 expression and activity via binding of free zinc, ultimately protected neurons from apoptosis. Meanwhile albumin treatment promoted activated microglia into multi-nucleated macrophagocytes and decreased the inflammation.ConclusionAlbumin alleviates esketamine-induced neuronal apoptosis through decreasing Zn2+ accumulation in GCL and downregulating Zn2+-dependent MMP9.

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes.

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe2+ chelating agent, 2,2’-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2’-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe2+. Together, our results show a fine balance between Fe2+-dependent enzymatic activity and Fe2+-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe2+, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns.

Iron-dependent epigenetic modulation promotes pathogenic T cell differentiation in lupus.

The trace element iron affects immune responses and vaccination, but knowledge of its role in autoimmune diseases is limited. Expansion of pathogenic T cells, especially T follicular helper (Tfh) cells, has great significance to systemic lupus erythematosus (SLE) pathogenesis. Here, we show an important role of iron in regulation of pathogenic T cell differentiation in SLE. We found that iron overload promoted Tfh cell expansion, proinflammatory cytokine secretion, and autoantibody production in lupus-prone mice. Mice treated with a high-iron diet exhibited an increased proportion of Tfh cell and antigen-specific GC response. Iron supplementation contributed to Tfh cell differentiation. In contrast, iron chelation inhibited Tfh cell differentiation. We demonstrated that the miR-21/BDH2 axis drove iron accumulation during Tfh cell differentiation and further promoted Fe2+-dependent TET enzyme activity and BCL6 gene demethylation. Thus, maintaining iron homeostasis might be critical for eliminating pathogenic Th cells and might help improve the management of patients with SLE.

Ppt1-deficiency dysregulates lysosomal Ca++ homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.

Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to infantile neuronal ceroid lipofuscinosis pathogenesis. Here, we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ transport from the endoplasmic reticulum to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4), which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease.

Evaluation of the influence of in vitro human digestion simulation on the chemical composition and bioactivities of Ziziphus jujuba Mill.

Abstract Fruit of Ziziphus jujuba Mill. are used as functional foods for centuries due to their rich content and bioactivities. Although in vitro antioxidant and hypoglycaemic activity of jujube fruit were investigated previously, the bioavailability phenomenon has been disregarded so far. For this study, 80% ethanol extract of Ziziphus jujuba fruit (ZJE) was investigated for its in vitro hypoglycaemic and antioxidant potentials, before and after the interaction with simulated human digestion. DPPH scavenging activity, FRAP, CUPRAC, and TOAC assays were used for this purpose. Moreover, inhibition potentials of α-amylase and α-glucosidase enzymes and advanced glycation end products (AGE) were examined for the hypoglycaemic effect. Results indicated that ZJE showed significant antioxidant and dose dependent enzyme and AGE inhibition activity. Nonetheless, subsequent to simulated human digestion in vitro bioactivities of ZJE were significantly lowered for bioavailable fraction (IN). Protocatechuic acid (PA) (major phenolic compound of the fruit) contents of the extract and fractions were measured via HPTLC for more accurate understanding of the effects of human digestion and bioavailability profile.

Effect of Detergents on Activity and Magnesium-Dependent Properties of Different Isoforms of Na+,K+-ATPase in the Crude Membrane Fraction of Rat Cerebral Cortex.

We studied the effect of various detergents (Tween-20, Triton X-100, and sodium deoxycholate) on activity and magnesium-dependent properties of Na+,K+-ATPase of the crude membrane fraction of rat cerebral cortex. All studied detergents significantly increased activity of the studied enzyme in a concentration-dependent manner. Sodium deoxycholate provided significantly higher values Na+,K+-ATPase activity (by ≈50%) than Triton X-100 and Tween-20. In the presence of Triton X-100, a changed pattern of the dependence of enzyme activity on the concentration of magnesium ions in the incubation solution was noted. Separate measurement of activities of Na+,K+-ATPase isoforms made it possible to assume that changes in magnesium-dependent properties are due to the predominant effect of Triton X-100 on ouabain-sensitive α2- and α3-isoforms.

The relation between acute changes in the systemic inflammatory response and circulating thiamine and magnesium concentrations after elective knee arthroplasty

Thiamine diphosphate (TDP) and magnesium are co-factors for key enzymes in human intermediary metabolism. However, their role in the systemic inflammatory response (SIR) is not clear. Therefore, the aim of the present study was to examine the relation between acute changes in the SIR and thiamine and magnesium dependent enzyme activity in patients undergoing elective knee arthroplasty (a standard reproducible surgical injury in apparently healthy individuals). Patients (n = 35) who underwent elective total knee arthroplasty had venous blood samples collected pre- and post-operatively for 3 days, for measurement of whole blood TDP, serum and erythrocyte magnesium, erythrocyte transketolase activity (ETKA), lactate dehydrogenase (LDH), glucose and lactate concentrations. Pre-operatively, TDP concentrations, erythrocyte magnesium concentrations, ETKA and plasma glucose were within normal limits for all patients. In contrast, 5 patients (14%) had low serum magnesium concentrations (< 0.75 mmol/L). On post-operative day1, both TDP concentrations (p < 0.001) and basal ETKA (p < 0.05) increased and serum magnesium concentrations decreased (p < 0.001). Erythrocyte magnesium concentrations correlated with serum magnesium concentrations (rs = 0.338, p < 0.05) and remained constant during SIR. Post-operatively 14 patients (40%) had low serum magnesium concentrations. On day1 serum magnesium concentrations were directly associated with LDH (p < 0.05), WCC (p < 0.05) and neutrophils (p < 0.01). Whole blood TDP and basal ETKA increased while serum magnesium concentrations decreased, indicating increased requirement for thiamine and magnesium dependent enzyme activity during SIR. Therefore, thiamine and magnesium represent potentially modifiable therapeutic targets that may modulate the host inflammatory response. Erythrocyte magnesium concentrations are likely to be reliable measures of status, whereas serum magnesium concentrations and whole blood TDP may not.ClinicalTrials.gov: NCT03554668.

Novel enzyme-based electrochemical and colorimetric biosensors for tetracycline monitoring in milk.

Recently, there has been a growing demand to develop portable devices for the fast detection of contaminants in food safety, healthcare, and environmental fields. Herein, two biosensing methods were designed by the use of nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent TetX2 enzyme activity and thionine as an excellent electrochemical and colorimetric mediator/probe to monitor tetracycline (TC) in milk. The nanoporous glassy carbon electrode (NPGCE) modified with polythionine was first prepared by electrochemically and then TetX2 was immobilized onto the NPGCE using polyethyleneimine. The prepared biosensor provided a high electrocatalytic response toward NAD(P)H by significantly reducing its overpotential. The proposed biosensor exhibited a detection limit of 40nM with a linear range of 0.1-0.8μM for TC determination. Besides, the thionine probe was used to develop a novel colorimetric assay using a simple enzymatic color reaction within a few minutes. The limit of detection for TC was experimentally achieved as 60nM, which was lower than the safety levels established by the World Health Organization (225nM). The correlation between change in the color of the solution and the concentration of TC was used for quality control of milk samples, as confirmed by the standard high-performance liquid chromatography method. The results show the great potential of the proposed assays as portable instruments for on-site TC measurements.

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

Centella asiatica alleviates diabetes-induced changes in fatty acid profile and oxidative damage in rat testis.

The in vivo effects of Centella asiatica L. Urban (Family: Apiaceae; CA) on diabetes-induced testicular fatty acid misdistribution and oxidative injury were investigated. Diabetic rats were treated with vehicle, CA or metformin daily for 14days by oral gavage. Fatty acid (FA) content in testis was analysed using gas chromatography-flame ionisation detection while redox indices were measured as peroxide value (PV), malondialdehyde (MDA), oxygen radical antioxidant capacity (ORAC), reduced glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. Diabetes increased omega-6 (61%), and decreased omega-3 (23%) and monounsaturated fatty acids (MUFA; 18%) compared to non-diabetic controls. Oxidative injury in diabetic rats was confirmed by increases in PV (112%) and MDA (77%) in addition to decreases in GSH (41%) and activities of GST (19%) & GPx (24%) compared to non-diabetic controls. CA treatment led to 17% reduction in omega-6 and 33% rise in MUFA compared to diabetic controls. Additionally, CA ameliorated the oxidative injury and improved antioxidant capacity by increasing GSH (49%), GST (16%) and GPx (23%) when compared to diabetic controls. Data suggest CA potential in alleviating the alterations caused by diabetes in testes through effects on omega-6 and MUFA; and via increased GSH level and dependent enzyme activities.

Amplified fluorescence of Mg2+ selective red-light emitting carbon dot in water and direct evaluation of creatine kinase activity

Carbon quantum dot/carbon dot (CD) exhibiting sustained photoluminescence at longer wavelengths in aqueous solution is difficult to prepare, but has enormous potential in biomedical applications. For the first time, we report the magnesium(II) selective fluorescence enhancement of a red-light emitting anthrarufin and boric acid-derived CD in aqueous solution for direct evaluation of creatine kinase (CK) enzyme activity. The CD displayed visually detectable, intense red fluorescence only in the presence of magnesium ion (Mg2+) at physiological pH value when irradiated with an ultraviolet (UV) source. Concurrently, a significant increase in steady-state fluorescence intensity and fluorescence lifetime was documented. A time-dependent density functional theory (TD-DFT) analysis displayed a bathochromic shift in UV-visible (vis) absorption, and increased oscillator strength of transition resulting from the selective chelation of Mg2+ with β-hydroxy keto functionality on the surface of the CD. The CD-Mg2+ assembly was subsequently used to conceptualize the detection of CK directly through the exploration of the differential binding affinity of Mg2+ with adenosine triphosphate (ATP), adenosine diphophate (ADP), and CD that is otherwise not possible with commercially available kits as of today. Thus, the report delineated here usher grandeur potential of CD for biological explorations related to Mg2+ or ATP sensing and monitoring of Mg2+-dependent enzymatic activity through a clear understanding of the chemistry.

Biochemical characterization and thermodynamic study of lipase from psychrotolerant Pseudomonas punonensis

Abstract A psychrotolerant lipolytic strain Pseudomonas punonensis was isolated from soil of cold desert of Leh (Laddakh) India. This strain was first time screened for production of extracellular lipase enzyme. The present study aimed for the production, partial purification, biochemical characterization, kinetic and thermodynamic parameters of lipase from this novel strain. Study of physico-chemical characteristic of lipase enzyme showed maximum activity at 30 °C and pH 9.0. Arrhenius plot of temperature dependent enzyme activity revealed the activation energy as 27.74 kJ/mol and the temperature quotient was calculated to be 1.01. The kinetic constants of the enzyme such as Vmax, and Km derived from Lineweaver- Burk plot of the enzyme activity recorded at 30 °C, were found to be 86.22 U mL−1 and 0.163 mM respectively. Experiment on thermal inactivation of enzyme showed thermal inactivation energy (Ea(d)) to be 56.89 kJ/mol. Thermodynamic values of lipase were found as Gibb's free energy (87.44 kJ/mol), enthalpy (54.29 kJ/mol) and entropy (-0.109 kJ/mol/K) for the enzyme denaturation obtained at 30 °C. The thermodynamic values suggested that enzyme catalysis was spontaneous and efficient at 30 °C when compared with 50 and 70 °C.

A biosensor for measuring NAD+ levels at the point of care

The cellular level of nicotinamide adenine dinucleotide (NAD+), through its different functions, affects cellular metabolism and signalling1-3. A decrease in the NAD+ content has been associated with various pathologies and physiological aging4,5, while strategies to boost cellular NAD+ levels have been shown to be effective against age-related diseases in many animal models6. The link between decreased NAD+ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD+, ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD+ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD+. This NAD+-dependent colour change enables the use of the biosensor in paper-based assays in which NAD+ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD+ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD+-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.

Functional consequences of enhanced expression of STIM1 and Orai1 in Huh-7 hepatocellular carcinoma tumor-initiating cells

BackgroundThe endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule1 (STIM1) activates the plasma membrane (PM) channel Orai1 in order to mediate store-operated Ca2+ entry (SOCE) in response to ER store depletion. Enhanced expression of STIM1 in cancer tissue has been associated with poor patient prognosis. Therefore, this study investigated the functional consequences of enhanced expression of STIM1 and Orai1 in a tumor-initiating subpopulation of Huh-7 hepatocellular carcinoma (HCC) cells that express epithelial cell adhesion molecule (EpCAM) and Prominin 1 (CD133).MethodsWe performed qRT-PCR, intracellular Ca2+ monitoring, protein analyses, and real-time cell proliferation assays on EpCAM(+)CD133(+) subpopulation of tumor-initiating Huh-7 HCC cells expressing high levels of STIM1 and/or Orai1. Statistical significance between the means of two groups was evaluated using unpaired Student’s t-test.ResultsEnhanced STIM1 expression significantly increased ER Ca2+ release and proliferation rate of EpCAM(+)CD133(+) cells.ConclusionSTIM1 overexpression may facilitate cancer cell survival by increasing ER Ca2+-buffering capacity, which makes more Ca2+ available for the cytosolic events, on the other hand, possibly preventing Ca2+-dependent enzymatic activity in mitochondria whose Ca2+ uniporter requires much higher cytosolic Ca2+ levels.

The nested enzyme-within-enterocyte (NEWE) turnover model for predicting dynamic drug and disease effects on the gut wall

Physiologically-based pharmacokinetic (PBPK) models provide a framework for in vitro-in vivo extrapolation of metabolic drug clearance. Many of the concepts in PBPK can have consequential impact on more mechanistic systems pharmacology models. In the gut wall, turnover of enzymes and enterocytes are typically lumped into one rate constant that describes the time dependent enzyme activity. This assumption may influence predictability of any sustained and dynamic effects such as mechanism-based inhibition (MBI), particularly when considering translation from healthy to gut disease.A novel multi-level systems PBPK model was developed. This model comprised a ‘nested enzyme-within enterocyte’ (NEWE) turnover model to describe levels of drug-metabolising enzymes. The ability of the model to predict gut metabolism following MBI and gut disease was investigated and compared to the conventional modelling approach.For MBI, the default NEWE model performed comparably to the conventional model. However, when drug-specific spatial crypt-villous absorption was considered, up to approximately 50% lower impact of MBI was simulated for substrates highly metabolised by cytochrome P450 (CYP) 3A4, interacting with potent inhibitors. Further, the model showed potential in predicting the disease effect of gastrointestinal mucositis and untreated coeliac disease when compared to indirect clinical pharmacokinetic parameters.Considering the added complexity of the NEWE model, it does not provide an attractive solution for improving upon MBI predictions in healthy individuals. However, nesting turnover may enable extrapolation to gut disease-drug interactions. The principle detailed herein may be useful for modelling drug interactions with cellular targets where turnover is significant enough to affect this process.

Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.