Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

Senolytic effects of quercetin in an in vitro model of pre-adipocytes and adipocytes induced senescence

The dysfunction of adipose tissue with aging and the accumulation of senescent cells has been implicated in the pathophysiology of chronic diseases. Recently interventions capable of reducing the burden of senescent cells and in particular the identification of a new class of drugs termed senolytics have been object of extensive investigation. We used an in vitro model of induced senescence by treating both pre-adipocytes as well as mature adipocytes with hydrogen peroxide (H2O2) at a sub-lethal concentration for 3 h for three consecutive days, and hereafter with 20 uM quercetin at a dose that in preliminary experiments resulted to be senolytic without cytotoxicity. H2O2 treated pre-adipocytes and adipocytes showed typical senescence-associated features including increased beta-galactosidase activity (SA-ß-gal) and p21, activation of ROS and increased expression of pro-inflammatory cytokines. The treatment with quercetin in senescent pre-adipocytes and adipocytes was associated to a significant decrease in the number of the SA-β-gal positive cells along with the suppression of ROS and of inflammatory cytokines. Besides, quercetin treatment decreased miR-155-5p expression in both models, with down-regulation of p65 and a trend toward an up-regulation of SIRT-1 in complete cell extracts. The senolytic compound quercetin could affect AT ageing by reducing senescence, induced in our in vitro model by oxidative stress. The downregulation of miRNA-155-5p, possibly through the modulation of NF-κB and SIRT-1, could have a key role in the effects of quercetin on both pre-adipocytes and adipocytes.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

TGF-β protects osteosarcoma cells from chemotherapeutic cytotoxicity in a SDH/HIF1α dependent manner

Background In the widespread adoption of chemotherapy, drug resistance has been the major obstacle to tumor elimination in cancer patients. Our aim was to explore the role of TGF-β in osteosarcoma-associated chemoresistance. Methods We performed a cytotoxicity analysis of methotrexate (MTX) and cisplatin (CIS) in TGF-β-treated osteosarcoma cells. Then, the metabolite profile of the core metabolic energy pathways in Saos-2 and MG-63 cell extracts was analyzed by 1H-NMR. We detected the expression of succinate dehydrogenase (SDH), STAT1, and hypoxia-inducible factor 1α (HIF1α) in TGF-β-treated osteosarcoma cells and further tested the effects of these molecules on the cytotoxicity induced by chemotherapeutic agents. Using in vivo experiments, we examined the tumor growth and survival time of Saos-2-bearing mice treated with a combination of chemotherapeutic agents and a HIF1α inhibitor. Results The metabolic analysis revealed enhanced succinate production in osteosarcoma cells after TGF-β treatment. We further found a decrease in SDH expression and an increase in HIF1α expression in TGF-β-treated osteosarcoma cells. Consistently, blockade of SDH efficiently enhanced the resistance of Saos-2 and MG-63 cells to MTX and CIS. Additionally, a HIF1α inhibitor significantly strengthened the anticancer efficacy of the chemotherapeutic drugs in mice with osteosarcoma cancer. Conclusion Our study demonstrated that TGF-β attenuated the expression of SDH by reducing the transcription factor STAT1. The reduction in SDH then caused the upregulation of HIF1α, thereby rerouting glucose metabolism and aggravating chemoresistance in osteosarcoma cells. Linking tumor cell metabolism to the formation of chemotherapy resistance, our study may guide the development of additional treatments for osteosarcoma.

Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.

HEXIM1 Is Essential for the Establishment of Appropriate Patterns of Gene Expression and Chromatin Architecture during Terminal Erythroid Maturation

Terminal erythroid maturation is associated with dramatic changes in gene expression in the setting of a cell that is undergoing rapid division and nuclear condensation. Disruption of this process is associated with inherited anemias and myelodysplastic syndromes. Recent work from our laboratory revealed that terminal erythroid maturation is associated with a dramatic decline in the level of total and elongation competent RNA polymerase II (Pol II), and that control of pol II activity is a critical step in the regulation of gene expression during terminal erythroid maturation. We further demonstrated that HEXIM1, which is highly expressed in early erythroid cells compared to most other cell types (biogps.org; bloodspot.eu), is essential for erythropoiesis (Murphy Blood 2021). The goal of our current study is to understand the mechanisms by which HEXIM1 regulates erythroid gene expression. HEXIM1 can impact gene expression though multiple mechanisms, most notably by associating with pTEFb, which is required for release of "paused" pol II into active transcription (reviewed in Michels, Transcription, 2018). HEXIM1 can inhibit transcription through sequestration of pTEFb in the 7SK ribonuclear complex, rendering it incapable of facilitating pause release. Alternatively, it can activate transcription by delivering pTEFb to target loci (McNamara Genome Data 2016). In erythroid cells, disruption of HEXIM1 impaired the expression of many erythroid specific genes, such as GYPA and many of the heme synthesis enzymes, while overexpression (OE) of HEXIM1 promoted their expression (Murphy, Blood, 2021). We therefore hypothesized that in maturing erythroblasts, HEXIM1 targets pTEFb to erythroid specific genes, promoting the establishment of appropriate patterns of gene expression and facilitating terminal erythroid maturation. To address this hypothesis, we generated novel HUDEP2 lines that OE HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb (Mbonye Proteomics 2015). Biotinylated 7SK pulldown confirmed that the Y271A mutation maintains the ability to bind the 7SK complex in erythroid cell extracts and RNA immunoprecipitation confirmed that the Y271A mutation increases the affinity of HEXIM1 for the 7SK complex in HUDEP2 cells. The Y271A mutation has significant functional consequences in erythroid cells. OE of wild type (WT) HEXIM1 in HUDEP2 cells resulted in enhanced proliferation in both expansion and maturation conditions, which was accompanied by increased cell and nuclear size, and a dramatic increase in the level of CD235a. Similar to our previously published HEXIM1 mutant with tyrosine to phenylalanine mutations at residues 271 and 274, the Y271A HEXIM1 mutation abrogated the enhanced proliferation seen with HEXIM1 OE in both expansion and maturation conditions. The Y271A mutation also rescued the larger cell and nuclear area associated with HEXIM1 OE, as well as the dramatic increase in the level of CD235a. Conversely, disruption of HEXIM1 via genome editing resulted in poor expansion and viability of HUDEP2 cells, which was rescued by expression of WT but not Y271A mutated HEXIM1, highlighting the importance of HEXIM1-pTEFb interactions for erythroid proliferation and survival. Further, OE of WT HEXIM1, but not the Y271A mutant, promoted erythroid gene expression while facilitating repression of genes that are normally silenced during terminal maturation, such as RPS19. In cells expressing WT HEXIM1 these gene expression changes were accompanied by increases in the global levels of ser2 and ser5 phosphorylated Pol II, as well as genome wide changes in their distribution. In contrast, the Y271A mutant decreased the global level of ser2 and ser5 pol II, consistent with its reduced ability to release pTEFb at target genes. Intriguingly, levels of H3K79me2, a histone mark reflective of active transcription through gene bodies, were decreased with OE of both WT and Y271A mutant HEXIM1, suggesting that the ability of HEXIM1 to promote transcriptional activation or repression is context dependent. Together, these data demonstrate a critical role for HEXIM1 and its interaction with pTEFb and the 7SK complex in the establishment of appropriate patterns of gene expression and chromatin architecture in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

The Toxicity Testing of Cyanobacterial Toxins In Vivo and In Vitro by Mouse Bioassay: A Review

: Different biological methods based on bioactivity are available to detect cyanotoxins, including neurotoxicity, immunological interactions, hepatotoxicity, cytotoxicity, and enzymatic activity. The mouse bioassay is the first test employed in laboratory cultures, cell extracts, and water bloom materials to detect toxins. It is also used as a traditional method to estimate the LD50. Concerning the ease of access and low cost, it is the most common method for this purpose. In this method, a sample is injected intraperitoneally into adult mice, and accordingly, they are assayed and monitored for about 24 hours for toxic symptoms. The toxin can be detected using this method from minutes to a few hours; its type, e.g., hepatotoxin, neurotoxin, etc., can also be determined. However, this method is nonspecific, fails to detect low amounts, and cannot distinguish between homologues. Although the mouse bioassay is gradually replaced with new chemical and immunological methods, it is still the main technique to detect the bioactivity and efficacy of cyanotoxins using LD50 determined based on the survival time of animals exposed to the toxin. In addition, some countries oppose animal use in toxicity studies. However, high cost, ethical considerations, low-sensitivity, non-specificity, and prolonged processes persuade researchers to employ chemical and functional analysis techniques. The qualitative and quantitative analyses, as well as high specificity and sensitivity, are among the advantages of cytotoxicity tests to investigate cyanotoxins. The present study aimed at reviewing the results obtained from in-vitro and in-vivo investigations of the mouse bioassay to detect cyanotoxins, including microcystins, cylindrospermopsin, saxitoxins, etc.

Identification and Deletion of The Genes Responsible for Hydrogen Production in Thermoanaerobacter Ethanolicus JW200

Background Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. Due to the coupling of hydrogenase activity with fermentation product distribution, understanding hydrogen production of T. ethanolicus, particularly the genes responsible, is valuable for metabolic engineering of the species. Results Utilizing the hydrogenases reported in Thermoanaerobacterium saccharolyticum and Pyrococcus furiosus as templates, BLAST search identified five hydrogenase gene clusters, including two membrane-bound [NiFe] hydrogenases ech and mbh, two cytoplasmic [FeFe] hydrogenases hyd and hydII, and one cytoplasmic [NiFe] hydrogenase shi. The combined deletion of ech, mbh, shi and hydG resulted in a strain that did not produce hydrogen and showed no methyl viologen hydrogenase activity in cell extracts. Strains with deletions of all the hydrogenases except one showed normal hydrogen production. Methyl viologen hydrogenase activity was greatly reduced in all combined deletion strains except the strain with an intact hydG gene. Conclusion High hydrogen production and hydrogenase activities have been observed for T. ethanolicus. Five hydrogenases have been identified. Hydrogen production was eliminated by deleting genes required for all five hydrogenases. Each individual hydrogenase was verified to be capable of producing hydrogen during fermentation, indicating a high degree of redundancy and flexibility in the hydrogenase systems of T. ethanolicus. A large portion of hydrogenase activity is encoded by the [Fe-Fe] hydrogenases.