Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.
Results
In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.
Conclusion
Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P1 from S1P to 18:1 LPA.
Results
We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P4. This mutation severely affected the response of S1P4 to S1P in a [35S]GTPγS binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P4 receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors.
Conclusions
Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P1 receptor.
We report on the synthesis of Fe3O4@SiO2 nanoparticles incorporated poly(divinylbenzene) monolithic column via in situ polymerization. The monolith had larger specific surface area and relatively uniform porous structure and was characterized by scanning electron microscopy, nitrogen adsorption–desorption, and Fourier transform infrared spectroscopy. The monolith was then applied for the evaluation of gas chromatography separation properties. Here, an electromagnetic induction heating technique was employed to control the column temperature with the thermal effect of eddy current in ferromagnetic materials Fe3O4. The monolith offered good separation efficiency for benzene and toluene and a higher column efficiency was obtained up to 4481 and 9216 plates per meter, respectively. In addition, the injection volume and column capacity of the proposed system are as much as 5 µL and 100 ng. This makes it possible to use a UV detector. The fabricated Fe3O4@SiO2 nanoparticles incorporated poly(divinylbenzene) monolithic column has been shown to be very promising for gas chromatography separation.
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.
Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.
Results
A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg-1 min-1 with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.
Conclusion
Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.
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.
Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.
Results
Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50?°C), thermal stability (50?°C) and Km (3.3?mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.
Conclusions
The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.
The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca2+ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca2+ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca2+ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca2+ cage within CR-domains from this family of receptors that function in endocytosis and signalling.
Results
We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca2+ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca2+, and show that CR56 binds 2 Ca2+ ions with an average affinity of KD = 10.6 microM, and there is no indication of additional Ca2+ binding sites within this receptor fragment.
Conclusions
Both CR-domains of CR56 bind a single Ca2+ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.
Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. Fusion is mediated by t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion proteins on the target membrane and v-SNAREs on the vesicle membrane; v-and t-SNAREs interact with each other, driving vesicle fusion with the target membrane. The Arabidopsis thaliana trans-Golgi network resident SNAREs SYP41 and VTI12, along with YKT61/62, have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in Arabidopsis cell extracts. Conflicting published results have indicated that SYP4 family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear.
Results
Here we used a proteoliposome fusion assay to demonstrate that SYP42 and SYP43 can substitute for SYP41 in driving lipid mixing, providing support for functional overlap between family members. Previous reports have also suggested that VTI11 and VTI12 SNAREs show partial overlap in function, despite having mostly distinct localizations and binding partners. We show that VTI11 can substitute for VTI12 in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo.
Conclusions
Our data provide biochemical evidence for functional overlap in membrane fusion between members of the SYP4 or VTI1 SNARE groups, supporting previous genetic data suggesting redundancy.
The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway.
Results
In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae.
Conclusions
The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.
Removal of ceftriaxone sodium antibiotic from water using cellulose acetate (CA) mixed matrix nanofiltration membranes was investigated in this work. Silica nanoparticles were functionalized with (3-aminopropyl)triethoxysilane (APTES). Then, the hydrophilic and negatively charged 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monomer was grafted from the surface of the amino-functionalized silica nanoparticles via surface-initiated redox polymerization. Finally, CA/silica and CA/modified silica nanocomposite membranes were prepared via phase inversion method, and the rejection of ceftriaxone sodium was studied. Rejection ratios were about 90 and 96% for CA/silica and CA/modified silica membranes at pH 8, respectively, wherein both were higher compared to the neat CA membrane. In fact, in case of nanocomposite membranes, size exclusion and charge repulsion between negatively charged functional groups of the membrane and anionic groups of drug operated synergistically at alkaline pH values, and the pharmaceutical rejection was improved.
NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.
Results
Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.
Conclusions
The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.
Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures.
Results
Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof.
Conclusion
Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.
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.
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.
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.
An increased incidence of fungal infections, both invasive and superficial, has been witnessed over the last two decades. Candida species seem to be the main etiology of nosocomial fungal infections worldwide with Candida albicans, which is commensal in healthy individuals, accounting for the majority of invasive Candida infections with about 30-40% of mortality.
Results
New aromatic and heterocyclic esters 5a-k of 1-aryl-3-(1H-imidazol-1-yl)propan-1-ols 4a-d were successfully synthesized and evaluated for their anti-Candida potential. Compound 5a emerged as the most active congener among the newly synthesized compounds 5a-k with MIC value of 0.0833 μmol/mL as compared with fluconazole (MIC value >1.6325 μmol/mL). Additionally, molecular modeling studies were conducted on a set of anti-Candida albicans compounds.
Conclusion
The newly synthesized esters 5a-k showed more potent anti-Candida activities than fluconazole. Compounds 7 and 8 revealed significant anti-Candida albicans activity and were able to effectively satisfy the proposed pharmacophore geometry, using the energy accessible conformers (Econf?<?20 kcal/mol).
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.
The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the “peroxide shunt” pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific “peroxygenation” catalysts.
Results
The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig’s blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25°C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044?±?0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150?±?0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction.
Conclusions
Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.
