Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.
Results
We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.
Conclusions
The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.
Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.
Methods
Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.
Results
In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.
Conclusion
With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.
The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens.
Methods
An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools.
Results
We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff’s stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75?±?0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively.
Conclusion
We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.
Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.
Methods
In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.
Results
In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.
Conclusions
The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.
The extracts from the aerial parts of Portulaca quadrifida have been reported to show the total flavonoid content, antioxidant and antibacterial activities.
Results
Our results revealed that the total flavonoid content of methanol and chloroform extracts is 2.335?±?0.0097 and 1.7312?±?0.0082 mgQE/100 g respectively. The two extracts also showed good antioxidant activity and total phenolic content as well as weak to moderate antibacterial activity against some bacteria.
Conclusions
The extracts the aerial parts of the P. quadrifida showed good total flavonoid content, DPPH radical scavenging activity and antibacterial activity. In addition to this, the extracts also showed the presence of some important compounds by phytochemical analysis.
Endonucleases play critical roles in maintaining genomic stability and regulating cell growth. The purpose of this study was to evaluate detection of endonuclease activity as an indicator in the early diagnosis and prognosis of lymphoma.
Results
The method of endonuclease activity determination was successfully established and applied to compare cancer patient and control cohorts. Endonuclease activities of cancer tissues were significantly higher than those of adjacent control tissues (P <?0.001). We next investigated endonuclease activity in peripheral blood of enrolled patients and the controls, which were also significantly higher in patients than in controls (P =?0.015). Additionally, endonuclease activities were elevated in the metastasis subgroup compared with the non-metastasis subgroup(P =?0.038), whereas no significant difference was found between age(≤ 56y, > 56y) and gender (P =?0.736?>?0.05 and P =?0.635?>?0.05, respectively). Although there was no significant difference between control group with the non-metastatic cancer patients (P =?0.800?>?0.05), endonuclease activities were lower in the control group compared with the non-metastatic cancer patients with lymphoma (P =?0.033). The progression-free survival probability of patients with elevated R ratios(R ratio?≥?1.4) was significantly lower than that of patients with lower R ratios (R ratio?<?1.4).
Conclusions
An assay was established to detect the endonuclease activity,which might be useful for the prognosis of cancers, especially lymphoma.
One of the most popular techniques for cancer detection is the nuclear medicine technique. The present research focuses on Platelet-12-lipoxygenase (P-12-LOX) as a promising target for treating and radio-imaging tumor tissues. Curcumin was reported to inhibit this enzyme via binding to its active site.
Results
A novel curcumin derivative was successfully synthesized and characterized with yield of 74%. It was radiolabeled with the diagnostic radioisotope technetium-99m with 84% radiochemical yield and in vitro stability up to 6 h. The biodistribution studies in tumor bearing mice confirmed the high affinity predicted by the docking results with a free binding energy value of (ΔG ?50.10 kcal/mol) and affinity (13.64 pki) showing high accumulation in solid tumor with target/non-target ratio >6.
Conclusion
The newly synthesized curcumin derivative, as a result of a computational study on platelet-12 lipoxygenase, showed its excellent free binding energy (?G ?50.10 kcal/mol) and high affinity (13.64 pKi). It could be an excellent radio-imaging agent that targeting tumor cells via targeting of P-12-LOX.
Graphical abstract This novel curcumin derivative was successfully synthesized and radiolabeled with technetium-99m and biologically evaluated in tumor bearing mice that showed high accumulation in solid tumor with target/non-target ratio >6 confirming the affinity predicted by the docking results. Predicted binding mode of a new curcumin derivative in complex with 12-LOX active site. b Curcumin itself in the 12-LOX active site biological distribution of 99mTc-curcumin derivative complex in solid tumor bearing Albino mice
The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).
Results
Here, we report the X-ray crystal structures of MtbL,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.
Conclusion
The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.
Quinoline 2-oxidoreductase (Qor) is a member of molybdenum hydroxylase which catalyzes the oxidation of quinoline (2, 3 benzopyridine) to 1-hydro-2-oxoquinoline. Qor has biological and medicinal significances. Qor is known to metabolize drugs produced from quinoline for the treatment of malaria, arthritis, and lupus for many years. However, the mechanistic action by which Qor oxidizes quinoline has not been investigated either experimentally or theoretically.
Purpose of the study
The present study was intended to determine the interaction site of quinoline, predict the transition state structure, and probe a plausible mechanistic route for the oxidative hydroxylation of quinoline in the reductive half-reaction active site of Qor.
Results
Density functional theory calculations have been carried out in order to understand the events taking place during the oxidative hydroxylation of quinoline in the reductive half-reaction active site of Qor. The most electropositivity and the lowest percentage contribution to the HOMO are shown at C2 of quinoline compared to the other carbon atoms. The transition state structure of quinoline bound to the active site has been confirmed by one imaginary negative frequency of ?104.500/s and ?1.2365899E+06 transition state energies. The Muliken atomic charges, the bond distances, and the bond order profiles were determined to characterize the transition state structure and the reaction mechanism.
Conclusion
The results have shown that C2 is the preferred locus of interaction of quinoline to interact with the active site of Qor. The transition state structure of quinoline bound to the active site has been confirmed by one imaginary negative frequency. Moreover, the presence of partial negative charges on hydrogen at the transitions state suggested hydride transfer. Similarly, results obtained from total energy, iconicity and molecular orbital analyses supported a concerted reaction mechanism.
The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.
Results
Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in KD value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.
Conclusions
Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.
The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies.
Results
The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC50 values of?<?20 μM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme.
Conclusions
In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.
Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.
Method
In this study, bone marrow cells and lung tissue cells of Balb/C were used as a source of hematopoietic stem cells and a source to production colony-stimulating factors, respectively. These cells were incubated with TDN separately and together.
Results
Briefly, the results of this study show that the effects of TDN has excitatory in concentrations lower than 50 µg/ml on colony formation and greater than 50 µg/ml is toxic to cells and it was inhibited the colony formation. Maximum stimulatory and inhibitory effects are shown in 50 and 400 µg/ml of crown ether and no colony was observed in the latter concentration.
Conclusion
The results from this study indicate that TDN significantly able to stimulate the colon formation while increased concentrations of TDN is inhibited colony formation by induction toxic effects due to excessive production of free radicals.
Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.
Results
The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.
Conclusions
AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.
Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ1-Δ2-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool.
Results
The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42?±?0.28 nkat mg * protein?1 and a kM value of 455?±?124 μM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C.
Conclusion
The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.
Novel six organic donor-π-acceptor molecules (D-π-A) used for Bulk Heterojunction organic solar cells (BHJ), based on thienopyrazine were studied by density functional theory (DFT) and time-dependent DFT (TD-DFT) approaches, to shed light on how the π-conjugation order influence the performance of the solar cells. The electron acceptor group was 2-cyanoacrylic for all compounds, whereas the electron donor unit was varied and the influence was investigated.
Methods
The TD-DFT method, combined with a hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP) in conjunction with a polarizable continuum model of salvation (PCM) together with a 6-31G(d,p) basis set, was used to predict the excitation energies, the absorption and the emission spectra of all molecules.
Results
The trend of the calculated HOMO–LUMO gaps nicely compares with the spectral data. In addition, the estimated values of the open-circuit photovoltage (Voc) for these compounds were presented in two cases/PC60BM and/PC71BM.
Conclusion
The study of structural, electronics and optical properties for these compounds could help to design more efficient functional photovoltaic organic materials.
In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined.
Results
The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34?±?0.96, 44.16?±?1.05, 65.45?±?1.29, 37.85?±?1.44 and 50.23?±?2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30?±?1.20, 53.80?±?1.07, 77.50?±?1.12, 26.30?±?1.35 and 37.78?±?1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC50?=?85 and 160 μg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques.
Conclusion
The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.
Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.
Methods
The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.
Results
We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.
Conclusion
This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.
Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery.
Results
Paired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery (controls) were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein (rhSIRPA) was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry (MS). Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected.
Conclusions
We conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.
