Β-galactosidase Enzyme Activity Research Articles

Biochar applications and enzyme activity, carbon dioxide emission, and carbon sequestration in a calcareous soil

Biochar is a carbon-rich product obtained by biomass pyrolysis and is considered as a means of carbon sequestration. However, there is limited knowledge regarding responses of soil respiration and C-cycle enzyme activities to BC in a calcareous soil. In this study, different biochar AS, TS, PP, CS were applied to the soil at different rates (0.5, 1.0, and 1.5%) The highest increase in soil organic C content was observed in the TS treatment, while the AS treatment caused a slight increase of soil organic C. The highest MBC content was recorded in AS treatment and the lowest was observed in TS. The highest CO2-C emission was recorded in the TS treatment. In contrast, the lowest CO2-C emission was observed in the PP treatment due to its high recalcitrant C content and was attributed to a positive priming effect, stabilizing BC mineralization to improve the soil. The highest β-galactosidase enzyme activity was observed in the CS treatment and the lowest activity was observed in AS. The highest change in β-glycosidase enzyme activity was observed of PP treatment and the lowest was in AS biochar application. Thus, TS treatment is recommended to increase the organic C content of soils, and the PP biochar can be used to reduce CO2-C emission. The TS, PP, and CS can be applied to increase enzyme activities. The study clearly shows that the addition of BC to arid soils may have a high potential to improve soil enzyme activities and subsequent carbon sequestration and biochemical cycles.

Visualized Enzyme-Activated Fluorescence Probe for Accurately Detecting β-Gal in Living Cells and BALB/c Nude Mice.

Colorectal cancer was one of the major malignant tumors threatening human health and β-Gal was recognized as a principal biomarker for primary colorectal cancer. Thus, designing specific and efficient quantitative detection methods for measuring β-Gal enzyme activity was of great clinical test significance. Herein, an ultrasensitive detection method based on Turn-on fluorescence probe (CS-βGal) was reported for visualizing the detection of exogenous and endogenous β-galactosidase enzyme activity. The test method possessed a series of excellent performances, such as a significant fluorescence enhancement (about 11.3-fold), high selectivity as well as superior sensitivity. Furthermore, under the optimal experimental conditions, a relatively low limit of detection down to 0.024 U/mL was achieved for fluorescence titration experiment. It was thanks to the better biocompatibility and low cytotoxicity, CS-βGal had been triumphantly employed to visual detect endogenous and exogenous β-Gal concentration variations in living cells with noteworthy anti-interference performance. More biologically significant was the fact that the application of CS-βGal in BALB/c nude mice was also achieved successfully for monitoring endogenous β-Gal enzyme activity.

Antibacterial activity of 2-hydroxy-4-methoxybenzaldehyde and its possible mechanism against Staphylococcus aureus.

Staphylococcus aureus causes several complicated infections. Despite decades of research on developing new antimicrobials, methicillin-resistant S. aureus (MRSA) remains a global health problem. Hence, there is a dire need to identify potent natural antibacterial compounds as an alternative to antimicrobials. In this light, the present work divulges the antibacterial efficacy and the action mechanism of 2-hydroxy-4-methoxybenzaldehyde (HMB) isolated from Hemidesmus indicus against S. aureus. Antimicrobial activity of HMB was assessed. HMB exhibited 1024µg ml-1 as the minimum inhibitory concentration (MIC) and 2×MIC as the minimum bactericidal concentration against S. aureus. The results were validated by spot assay, time kill, and growth curve analysis. In addition, HMB treatment increased the release of intracellular proteins and nucleic acid contents from MRSA. Additional experiments assessing the structural morphology of bacterial cells using SEM analysis, β-galactosidase enzyme activity, and the fluorescence intensities of propidium iodide and rhodamine123 dye divulged that the cell membrane as one of the targets of HMB to hinder S. aureus growth. Moreover, the mature biofilm eradication assay revealed that HMB dislodged nearly 80% of the preformed biofilms of MRSA at the tested concentrations. Further, HMB treatment was found to sensitize MRSA cells upon combining tetracycline treatment. The present study suggests that HMB is a promising compound with antibacterial and antibiofilm activities and could act as a lead structure for developing new antibacterial drugs against MRSA.

Enzyme activity of β-galactosidase immobilized in polyelectrolyte multilayers deposited on corona charged composite substrates

In the present paper chitosan and xanthan polyelectrolyte multilayers (PEMs) deposited on composite multicomponent poly(D-lactic acid) (PDLA) / poly(ε-caprolactone) (PEC) substrates were investigated. The substrates' melting phenomena and degree of crystallinity were investigated by differential scanning calorimetry (DSC). Based on the melting behaviour it was suggested that PLA and PEC are not miscible at molecular level and no co-crystallization is realized. The substrates obtained were charged in a corona discharge. Positive or negative 5 kV voltages were applied to the corona electrode and 1 kV voltage of the same polarity - to the grid. The normalized surface potential dependences on the storage times were investigated. The results obtained show that the values of the normalized surface potential decay faster for negatively charged substrates. Layer-by-layer (LbL) technique was used for multilayer deposition on the substrates. PEMs with 8 layers were obtained. The enzyme of β-galactosidase was immobilized in the obtained polyelectrolyte multilayers. The enzyme activity of the immobilized enzyme was studied. The experimental results obtained show that the enzyme immobilization in positively charged films have higher activity compared to the negatively charged ones, independently of substrates type.

Camellia Seed Cake Extract Supports Hair Growth by Abrogating the Effect of Dihydrotestosterone in Cultured Human Dermal Papilla Cells.

Autocrine and paracrine factors play key roles in the process of Androgenetic alopecia (AGA), which are secreted by balding dermal papilla cells (DPCs) after dihydrotestosterone (DHT) induction. Camellia seed cake is an oriental oil extraction byproduct, and its extract has been traditionally used to wash hair in China. This study elucidated the hair growth-promoting effects of Camellia seed cake extract (CSCE) in DHT-treated cultured DPCs and its underlying mechanisms. The effect of CSCE on cell viability and release of inflammatory factors IL-6 and IL-1α was performed on human dermal papilla cells (DPCs) incubated with DHT. Relative expression of bax, bcl-2, p53, androgen receptor (AR) and 5α- reductase type II (SRD5A2) was determined by PCR. Senescence-associated was examined by β-galactosidase (SA-β-Gal) assays. CSCE restored DHT-induced cell damage in a dose-dependent manner, and effectively reduced the production of IL-6 and IL-1α in DHT-treated DPCs. CSCE exhibited an anti-apoptotic effect, which increased the expression of bcl-2, and decreased the expressions of bax and p53 in DHT-incubated DPCs. CSCE also showed an anti-androgenic effect reversing the increase in AR and SRD5A2 expressions in DPCs driven by DHT incubation. In addition, CSCE inhibited the β-galactosidase enzyme activity and slowed down the cell senescence of DPCs which is crucial for AGA progression. In this study, we found that CSCE may have the potential to prevent and alleviate AGA by abrogating the effect of DHT in cultured DPCs.

Summary of Probiotic Potential of the Kefir for the Prevention and Alleviation of Functional GI Diseases

Background: Functional Gastrointestinal (GI) disease is a term that refers to a range of disease conditions involving chronic diarrhea, irritable bowel syndrome, constipation, inflammatory bowel disease (IBD), and lactose intolerance. Statistical reports have documented >40% of the world's population is being afflicted by functional GI disorders. Consequences of frequent intake of antibiotics include mortality of gut microflora, antibiotic-associated diarrhea, the establishment of antibiotic resistance, and inference with healthy bodily functions. To overcome the harms of antibiotics, studies are being conducted to determine the potential of probiotics as alternatives to antibiotics. Aim of Study: The purpose of constructing this literature review is to outline the potential attributes of Kefir and to assess the solitary efficiency of this beverage in the amelioration of GI disease conditions. Methodology: Extraction of data entailed digging of literature hubs including PubMed, PubMed Central, Research Gate, Science Direct, and Google Scholar, employing a different combination of keywords to retrieve substantial details. Selection criteria of the articles involved the published reports of preceding 10 years (2012-2021) that exhibited relevancy with the topic Conclusion: Kefir grains having different origins provide a distinct array of antimicrobial spectrums and are found to be effective against different pathogens. IBDs, diarrheal sicknesses, and constipation can be alleviated by the consumption of this beverage. Moreover, the probioticantibiotic combination is also essential for the mitigation of several diseases. The synergistic impact of several microbial strains is vital for the treatment of celiac disease. Lactose maldigestion can be relieved by the consumption of kefir having β-galactosidase enzyme activity.

Hematopoietic stem cell gene therapy ameliorates CNS involvement in murine model of GM1-gangliosidosis

GM1-gangliosidosis is a progressive neurodegenerative glycosphingolipidosis resulting from a GLB1 gene mutation causing a deficiency of the lysosomal enzyme β-galactosidase, which leads to the abnormal accumulation of GM1 ganglioside in the central nervous system. In the most severe early infantile phenotype, excessive ganglioside accumulation results in a rapid decline in neurological and psychomotor functions, and death occurs within 2 years of age. Currently, there is no effective therapy for GM1-gangliosidosis. In this study, we evaluated the therapeutic efficacy of ex vivo gene therapy targeting hematopoietic stem cells using a lentiviral vector to increase enzyme activity, reduce substrate accumulation, and improve astrocytosis and motor function. Transplanting GLB1-transduced hematopoietic stem cells in mice increased β-galactosidase enzyme activity in the central nervous system and visceral organs. Specifically, this gene therapy significantly decreased GM1 ganglioside levels in the brain, especially in the cerebrum. More important, this gene therapy rectified astrocytosis in the cerebrum and improved motor function deficits. Furthermore, the elevation of serum β-galactosidase activity in secondary-transplanted mice suggested the ability of transduced hematopoietic stem cells to repopulate long term. These data indicate that ex vivo gene therapy with lentiviral vectors is a promising approach for the treatment of brain deficits in GM1 gangliosidosis.

Biomarkers of ageing in the study of occupational harm impacts (literature review)

Aging is an individual, complex biological process, modulated by internal and external factors, characterized by a progressive loss of biological / physiological integrity, which leads to body dysfunction, increases vulnerability and death. Influence of activity type on aging rate has been convincingly shown in many studies, which makes it possible assess differences in aging rate of workers, exposed various occupational factors, conditions, work nature and intensity in certain professional and seniority groups, adequately reflects health state and can predict effectiveness of human labor activity. As integral indicator, it can help identify individuals at risk of age-related disorders, serving as a measure of relative fitness and predicting later life disability and mortality, regardless of chronological age. The article provides an overview of the main measuring ageing rate methods based on biomarkers, such as functional (“Kiev model”, WAI) and molecular genetic biomarkers (determination of telomere length, β-galactosidase enzyme activity) of human ageing, applicable in occupational medicine. The review discusses the main requirements for biomarker sets compilation, methods applicability and reliability, mathematical approaches to biological age calculating, and some workers biological age calculating problems. This allows assuming the great potential for using biological age to assess the impact of working conditions and work nature on workers’ ageing rate to prevent disability and improve quality of life.

Comparative studies of the biological efficacies of Ag and Ag-MgO nanocomposite formed by the green synthesis route

The present study investigates the changes occurring to the physicochemical and biological properties of silver (Ag) nanoparticles (NPs) before and after conjugating with that of magnesium oxide (MgO). In that view, to form Ag NPs and Ag-MgO nanocomposite, we adopted a green synthesis route that makes use of Citrus paradisi(grapefruit red) extract as a reducing agent and the formed particles are thoroughly characterized for the crystallinity, surface bonding and functionality, morphology, composition, surface charges, etc. From the physicochemical analysis, the successful formation of Ag and Ag-MgO composites are confirmed from the UV–Vis and FTIR spectroscopy, while the TEM indicated the spherical morphology and DLS for the average particle size of 260 nm in solution. Further, on testing the biological efficacy, the Ag-MgO nanocomposite (up to 320 µg/mL) was found to have efficient antibacterial and antifungal activities against Staphylococcus aureus and Candida albicans (respectively). Also, the studies of β-galactosidase enzyme activity resulted that Ag’s composite formation with MgO (Ag-MgO with IC50 of 27.6 µg/mL) is not significantly enhancing the effectiveness as compared to pure Ag NPs (IC50 of 28.6 µg/mL). Also, the in vitro cell viability studies indicated the anticancer activity of Ag-MgO nanocomposite in a dose-dependent manner when tested against the Hela cancer cells was in the concentration range of 1–100 µg/mL (24 h period). Based on the overall analysis, the easy making process by the green synthesis route and inexpensive Ag-MgO nanocomposite can serve as a superior antimicrobial and anticancer ingredient for the development of therapeutic products in the biomedical sector.

High-Throughput Virtual Screening for a New Class of Antagonist Targeting LasR of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes fatal effects in patients with cystic fibrosis and immunocompromised individuals and leads to around 1000 deaths annually. The quorum sensing mechanism of P. aeruginosa plays a major role in promoting biofilm formation and expression of virulent genes. Hence, quorum sensing inhibition is a promising novel approach to treat these bacterial infections as these organisms show a wide range of antibiotic resistance. Among the interconnected quorum sensing network of P. aeruginosa, targeting the las system is of increased interest as its principal receptor protein LasR is the earliest activated gene. It is also shown to be involved in the regulation of other virulence-associated genes. In this study, we have applied high-throughput virtual screening, an in silico computational method to identify a new class of LasR inhibitors that could serve as potent antagonists to treat P. aeruginosa-associated infections. Three-tire structure-based virtual screening was performed on the Schrödinger small molecule database, which resulted in 12 top hit compounds with docking scores lesser than −11.0 kcal/mol. Three of these best-scored compounds CACPD2011a-0001928786 (C1), CACPD2011a-0001927437 (C2), and CACPD2011a-0000896051 (C3) were further analyzed. The binding free energies of these compounds in complex with the target protein LasR (3IX4) were evaluated, and the pharmacokinetic properties were determined. The stability of the docked complexes was assessed by running a molecular dynamics simulation for 100 ns. Molecular dynamics simulation analysis revealed that all three compounds were found to be in stable contact with the protein over the entire simulation period. The antagonistic effect of these compounds was validated using the LasR reporter gene assay in the presence of acyl homoserine lactone. Significant reduction in the β-galactosidase enzyme activity was achieved at 100 nM concentration for all three compounds pursued. Hence, the present study provides strong evidence that these three compounds could serve as quorum sensing inhibitors of P. aeruginosa LasR protein and can be a probable candidate to treat Pseudomonas-associated infections.

Supramolecular Interaction of Molecular Cage and β‐Galactosidase: Application in Enzymatic Inhibition, Drug Delivery and Antimicrobial Activity

Enzyme inhibitors play a crucial role in diagnosis of a wide spectrum of diseases related to bacterial infections. We report here the effect of a water-soluble self-assembled PdII 8 molecular cage towards β-galactosidase enzyme activity. The molecular cage is composed of a tetrapyridyl donor (L) and cis-[(en)Pd(NO3 )2 ] (en=ethane-1,2-diamine) acceptor and it has a hydrophobic internal cavity. We have observed that the acceptor moiety mainly possesses the ability to inactivate the β-galactosidase enzyme activity. Kinetic investigation revealed the mixed mode of inhibition. This inhibition strategy was extended to control the growth of methicillin-resistant Staphylococcus aureus. The internalization of the Pd(II) cage inside the bacteria was confirmed when bacterial solutions were incubated with curcumin loaded cage. The intrinsic green fluorescence of curcumin made the bacteria glow when put under an optical microscope. Furthermore, this curcumin loaded molecular cage shows an enhanced antibacterial activity. Thus, PdII 8 molecular cage is quite attractive due to its dual role as enzyme inhibitor and drug carrier.

A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy.

GM1 gangliosidosis is a rare lysosomal storage disease caused by a deficiency of β-galactosidase due to mutations in the GLB1 gene. We established a C57BL/6 mouse model with Glb1G455R mutation using CRISPR/Cas9 genome editing. The β-galactosidase enzyme activity of Glb1G455R mice measured by fluorometric assay was negligible throughout the whole body. Mutant mice displayed no marked phenotype at eight weeks. After 16 weeks, GM1 ganglioside accumulation in the brain of mutant mice was observed by immunohistochemical staining. Meanwhile, a declining performance in behavioral tests was observed among mutant mice from 16 to 32 weeks. As the disease progressed, the neurological symptoms of mutant mice worsened, and they then succumbed to the disease by 47 weeks of age. We also observed microglia activation and proliferation in the cerebral cortex of mutant mice at 16 and 32 weeks. In these activated microglia, the level of autophagy regulator LC3 was up-regulated but the mRNA level of LC3 was normal. In conclusion, we developed a novel murine model that mimicked the chronic phenotype of human GM1. This Glb1G455R murine model is a practical in vivo model for studying the pathogenesis of GM1 gangliosidosis and exploring potential therapies.

Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production

Abstract. Indah AWN, Rohmatussolihat, Rahayu WP, Setyoningrum F, Priadi G, Afianti F. 2020. Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production. Biodiversitas 21: 5403-5407. Deficiency of β-galactosidase enzyme causes lactose to become undigested in gastrointestinal system, therefore the system needs further addition of external β-galactosidase. The sources β-galactosidase enzyme varies from plants, animals, and microorganisms. In industrial applications, microorganisms have become a considered potential source of β-galactosidase. Lactobacillus brevis LIPI13-2-LAB131 had high β-galactosidase enzyme activity, which was 7.93 U/mL. The aim of this research was to optimize the growth condition of L. brevis LIPI13-2-LAB131 in order to produce maximum β-galactosidase enzyme activity. This research consisted of performing optimization processes using design expert 7.0 (DX7) program with response surface methodology (RSM) and partial purification of β-galactosidase enzyme. The results of this research showed that the optimum growth conditions of L. brevis LIPI13-2-LAB131 were in 1.48% lactose level, incubation temperature of 34.91 °C, incubation time of 48.48 hours, and 2.83% inoculum size with desirability value of 0.839. The result of enzyme purification showed that value of β-galactosidase enzyme activity increased up to 22.88 ± 0.29 U/mL with purification yield of 11.65%.

Mesenchymal Stem Cells Promote Caspase Expression in Molt-4 Leukemia Cells Via GSK-3α/Β and ERK1/2 Signaling Pathways as a Therapeutic Strategy.

Mesenchymal stem cells (MSCs) are considered an interesting tool in cell therapy due to their unique features such as self-renewal, multi-potency, and pluripotency. The multifunctional properties of these cells are being investigated in many studies. The current research examined the influence of MSCs on the Molt-4 cell line as acute lymphoblastic leukemia (ALL) cells. MSCs were cultured, characterized, and co-cultured with Molt-4 cells in a trans-well system. Then, cultured Molt-4 alone and Molt-4 co-cultured with MSCs (10:1) were collected on day 7 and subjected to western blotting for protein expression assessment. Telomerase activity as well as cell senescence, were investigated by PCR-ELISA TRAP assay and β-galactosidase activity measurement, respectively. It was found that MSCs resulted in a significant increase in the pro-caspase-8 and cleaved-caspase 8 and 9 expression levels. Furthermore, protein expression levels of GSK-3α/β and ERK1/2 were significantly decreased. The results also showed that MSCs caused significant decreases and increases in telomerase and β-galactosidase enzyme activity of Molt-4 cells, respectively. It was concluded that MSCs co-cultured with Molt-4 cells could be involved in the promotion of Molt-4 cell apoptosis and cell senescence via caspase-8, 9 cascade and GSK-3α/β and ERK1/2 signaling pathways.

Dynamics of Matrix Metalloproteinase Activity and Extracellular Matrix Proteins Content in the Process of Replicative Senescence of Human Mesenchymal Stem Cells

A comparative analysis of mesenchymal stem cells (MSCs) of differing origin is important because of their specific interaction with a unique microenvironment (niche) in a particular tissue. Some cellular processes are regulated through the interaction of extracellular matrix (ECM) proteins with matrix metalloproteinases (MMPs). In this work, we compared the dynamics of MMP activity and the level of ECM proteins during replicative senescence of three lines of human MSCs obtained from Wharton’s jelly of human umbilical cord (MSCWJ-1 line), eyelid skin (DF-2 line), and human epicardial adipose tissue isolated during coronary artery bypass grafting (ADH-MSC line). The fractions of cells with β-galactosidase enzyme activity (marker of replicative senescence) and the content of ECM proteins (fibronectin and type I collagen), as well as the activity of MMP-1, MMP-2, and MMP-9, were analyzed during long-term cultivation. It was found that three lines differ in the content of fibronectin, type 1 collagen, and MMP activity during the replicative senescence. Cells of the ADH-MSC line are mostly different from the other two lines in terms of aging rate, ECM protein content, and MMP activity. The reason for this discrepancy is that the cells are obtained from the tissue of a patient having heart disease.

Development of Enzymatic Microtoxicity Test for the Toxicity Assay of Chlorophenolic Compounds

Enzymes are proteins that serve as catalysts of biological reactions in the animal, plant, and microbial cells. They are quite specific concerning with regard to their substrates. Enzymes lower the activation energy and increase the rate of biochemical reactions. According to the type of reaction catalyzed, enzymes are currently subdivided into six classes, namely oxidoreductases, hydrolases, transferases, lyases, isomerases, and ligases.
 The effects of toxic chemicals and chlorophenolic compounds on enzyme activity are determined by fast, useful, and simple enzymatic methods. These methods can be carried out in low volumes and automatically using equipment such as a spectrophotometer, fluorometer and scintillation counters.
 In this study, a new microtoxicity test was developed with the use of the fungal and bacterial β-galactosidase enzyme. In the toxicity experiments, the effects of 2,4,6-trichlorophenol (2,4,6 TCP), 2,4-dichlorophenol (2,4 DCP) and 4-chlorophenol (4 CP) over the β-galactosidase enzyme activity were investigated.
 In this study, the bacterial and fungal β-galactosidase enzyme was found to be effective in determining the detoxification of some chlorinated phenolic compounds, 2,4,6-TCP, 2,4-DCP, and 4-CP, through the treatment with laccase enzyme. The inhibitor effect of toxic compounds on the activity of the β-galactosidase enzyme was examined. As a result of the studies, 61% inhibition for 2,4,6-TCP, 58% inhibition for 2,4-DCP and 47% inhibition for 4-CP was observed.
 Mikrotoxicity test system developed in our laboratory which works principally based on inactivation of β-galactosidase activity by toxic chlorinated phenolics, was used for toxicity detection. Data obtained from microtoxicity tests have shown that the dechlorination of chlorinated phenolics with laccase has resulted in the loss of toxic potentiality of these chemicals to a substantial extent.
 We thought that this study is to be an important contribution to bioremediation science and environmental technology with a new biotechnological approach.

Effect of blue light at 410 and 455 nm on Pseudomonas aeruginosa biofilm

Pseudomonas aeruginosa is an opportunistic pathogen resistant to many antibiotics, able to form biofilm and causes serious nosocomial infections. Among anti-Pseudomonas light-based approaches, the recent antimicrobial Blue Light (aBL) treatment seems very promising.The aim of this study was to evaluate the efficiency of blue light in inhibiting and/or eradicating P. aeruginosa biofilm. Light at 410 nm has been identified as successful in inhibiting biofilm formation not only of the model strain PAO1, but also of CAUTI (catheter-associated urinary tract infection) isolates characterized by their ability to form biofilm. Results of this work on 410 nm light also demonstrated that: i) at the lowest tested radiant exposure (75 J cm−2) prevents matrix formation; ii) higher radiant exposures (225 and 450 J cm−2) light impairs the cellular components of biofilm, adherent and planktonic ones; iii) light eradicates with a good rate young and older biofilms in a light dose dependent manner; iv) it is also efficient in inactivating catalase A, a virulence factor playing an important role in pathogenic mechanisms.Light at 455 nm, even if at a lower extent than 410 nm, showed a certain anti-Pseudomonas activity. Furthermore, light at 410 nm caused detrimental effects on enzyme activity of β-galactosidase and catalase A, and changes on plasmid DNA conformation and ortho-nitrophenyl-β-D-galactopyranoside structure.This study supports the potential of blue light for anti-infective and disinfection applications.

Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

A β-galactosidase-expressing E. coli culture as an alternative test to identify skin sensitizers and non-sensitizers

ABSTRACTAlthough the Organization for Economic Cooperation and Development (OECD) has adopted several in vitro methods with reasonable predictive capacity, alternative methods for identifying skin sensitizers and non-sensitizers with reliability and simplicity are still required for more efficient and economic prediction. The present study was to design an in vitro system with the use of a β-galactosidase-expressing E. coli culture for simpler but sufficiently accurate classification of skin sensitizers and non-sensitizers. A LacZ gene-containing E. coli strain that is capable of producing β-galactosidase enzyme was induced by isopropyl β-D-1-thiogalactopyranoside with concomitant treatment with test chemicals. After 6-hr incubation, cells were lysed and β-galactosidase enzyme activity was monitored colorimetrically by using O-nitrophenyl-D-galactopyranoside as a substrate. Following optimization of several experimental conditions, 22 skin sensitizers and 11 non-sensitizers were examined to assess predictive capacity of this method. The results indicated that predictivity was as follows: 90.9% sensitivity, 81.8% specificity, and 87.9% accuracy, when 17.3% of control activity was used as the cut-off value to separate sensitizers from non-sensitizers. Data suggested that the current bacterial system expressing β-galactosidase may serve as a useful alternative test for classifying skin sensitizers and non-sensitizers, without the utilization of animals or mammalian cell cultures.

Dendrimer-like nanoparticles based β-galactosidase assembly for enhancing its selectivity toward transgalactosylation

Functional nanomaterials have been pursued to assemble nanobiocatalysts since they can provide unique hierarchical nanostructures and localized nanoenvironments for enhancing enzyme specificity, stability and selectivity. Functionalized dendrimer-like hierarchically porous silica nanoparticles (HPSNs) was fabricated for assembling β-galactosidase nanobiocatalysts for bioconversion of lactose to galacto-oligosaccharides (GOS). The nanocarrier was functionalized with amino (NH2) and carboxyl (COOH) groups to facilitate enzyme binding, benchmarking with non-functionalized HPSNs. Successful conjugation of the functional groups was confirmed by FTIR, TGA and zeta potential analysis. HPSNs-NH2 showed 1.8-fold and 1.1-fold higher β-galactosidase adsorption than HPSNs-COOH and HPSNs carriers, respectively, with the highest enzyme adsorption capacity of 328mg/g nanocarrier at an initial enzyme concentration of 8mg/ml. The HPSNs-NH2 and β-galactosidase assembly (HPSNs-NH2-Gal) demonstrated to maintain the highest activity at all tested enzyme concentrations and exhibited activity up to 10 continuous cycles. Importantly, HPSNs-NH2-Gal was simply recycled through centrifugation, overcoming the challenging problems of separating the nanocarrier from the reaction medium. HPSNs-NH2-Gal had distinguished catalytic reaction profiles by favoring transgalactosylation, enhancing GOS production of up to 122g/l in comparison with 56g/l by free β-galactosidase. Furthermore, it generated up to 46g/l GOS at a lower initial lactose concentration while the free counterpart had negligible GOS production as hydrolysis was overwhelmingly dominant in the reaction system. Our research findings show the amino-functionalized HPSNs can selectively promote the enzyme activity of β-galactosidase for transgalactosylation, which is beneficial for GOS production.