scholarly journals Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor

1997 ◽  
Vol 327 (3) ◽  
pp. 643-650 ◽  
Author(s):  
Laurence DINAN ◽  
Pensri WHITING ◽  
Jean-Pierre GIRAULT ◽  
René LAFONT ◽  
S. Tarlochan DHADIALLA ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Antagonistic Activity ◽  
Side Chain ◽  
Ecdysteroid Receptor ◽  
Whole Cells ◽  
Definitive Evidence ◽  
Permanent Cell ◽  
A Cell ◽  
Gel Shift Assay ◽  
Hormone Antagonists

Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 μM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 μM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Δ23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.

Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

1968 ◽  
Vol 14 (1) ◽  
pp. 25-31 ◽  
Author(s):  
G. W. Strandberg ◽  
P. W. Wilson
Keyword(s):  
Color Change ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Color Difference ◽  
Potassium Nitrate ◽  
Whole Cells ◽  
System A ◽  
Growth System ◽  
A Cell ◽  
Enzyme Formation

The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be due to a color change in the cells from reddish brown to dark brown. Spectrophotometric examination of extracts from ammonia- and N2-grown cells did not reveal any components responsible for this color difference, but this result may reflect only the presence of interfering substances in the crude extract.

Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents.

1996 ◽  
Vol 40 (2) ◽  
pp. 443-447 ◽  
Author(s):  
B Favre ◽  
N S Ryder
Keyword(s):  
Biochemical Characterization ◽  
Trichophyton Rubrum ◽  
Side Chain ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
High Potency ◽  
Whole Cells ◽  
Absolute Requirement ◽  
Tertiary Amino

Squalene epoxidase (SE) is the primary target of the allylamine antimycotic agents terbinafine and naftifine and also of the thiocarbamates. Although all of these drugs are employed primarily in dermatological therapy, SE from dermatophyte fungi has not been previously investigated. We report here the biochemical characterization of SE activity from Trichophyton rubrum and the effects of terbinafine and other inhibitors. Microsomal SE activity from T. rubrum was not dependent on soluble cytoplasmic factors but had an absolute requirement for NADPH or NADH and was stimulated by flavin adenine dinucleotide. Kinetic analyses revealed that under optimal conditions the Km for squalene was 13 microM and its Vmax was 0.71 nmol/h/mg of protein. Terbinafine was the most potent inhibitor tested, with a 50% inhibitory concentration (IC50) of 15.8 nM. This inhibition was noncompetitive with regard to the substrate squalene. A structure-activity relationship study with some analogs of terbinafine indicated that the tertiary amino structure of terbinafine was crucial for its high potency, as well as the tert-alkyl side chain. Naftifine had a lower potency (IC50, 114.6 nM) than terbinafine. Inhibition was also demonstrated by the thiocarbamates tolciclate (IC50, 28.0 nM) and tolnaftate (IC50, 51.5 nM). Interestingly, the morpholine amorolfine also displayed a weak but significant effect (IC50, 30 microM). T. rubrum SE was only slightly more sensitive (approximately twofold) to terbinafine inhibition than was the Candida albicans enzyme. Therefore, this difference cannot fully explain the much higher susceptibility (> or = 100-fold) of dermatophytes than of yeasts to this drug. The sensitivity to terbinafine of ergosterol biosynthesis in whole cells of T. rubrum (IC50, 1.5 nM) is 10-fold higher than that of SE activity, suggesting that the drug accumulates in the fungus.

scholarly journals Anti-Carcinogenic Glucosinolates in Cruciferous Vegetables and Their Antagonistic Effects on Prevention of Cancers

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2983 ◽  
Author(s):  
Prabhakaran Soundararajan ◽  
Jung Kim
Keyword(s):  
Antagonistic Activity ◽  
Antagonistic Effect ◽  
Side Chain ◽  
Cruciferous Vegetables ◽  
Related Factor ◽  
Nuclear Factor ◽  
Naturally Occurring ◽  
Therapeutic Properties ◽  
Responsive Element

Glucosinolates (GSL) are naturally occurring β-d-thioglucosides found across the cruciferous vegetables. Core structure formation and side-chain modifications lead to the synthesis of more than 200 types of GSLs in Brassicaceae. Isothiocyanates (ITCs) are chemoprotectives produced as the hydrolyzed product of GSLs by enzyme myrosinase. Benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane ([1-isothioyanato-4-(methyl-sulfinyl) butane], SFN) are potential ITCs with efficient therapeutic properties. Beneficial role of BITC, PEITC and SFN was widely studied against various cancers such as breast, brain, blood, bone, colon, gastric, liver, lung, oral, pancreatic, prostate and so forth. Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor limits the tumor progression. Induction of ARE (antioxidant responsive element) and ROS (reactive oxygen species) mediated pathway by Nrf2 controls the activity of nuclear factor-kappaB (NF-κB). NF-κB has a double edged role in the immune system. NF-κB induced during inflammatory is essential for an acute immune process. Meanwhile, hyper activation of NF-κB transcription factors was witnessed in the tumor cells. Antagonistic activity of BITC, PEITC and SFN against cancer was related with the direct/indirect interaction with Nrf2 and NF-κB protein. All three ITCs able to disrupts Nrf2-Keap1 complex and translocate Nrf2 into the nucleus. BITC have the affinity to inhibit the NF-κB than SFN due to the presence of additional benzyl structure. This review will give the overview on chemo preventive of ITCs against several types of cancer cell lines. We have also discussed the molecular interaction(s) of the antagonistic effect of BITC, PEITC and SFN with Nrf2 and NF-κB to prevent cancer.

scholarly journals Methods and applications of label-free cell-based systems

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Joachim Wiest
Keyword(s):  
Environmental Monitoring ◽  
Morphological Changes ◽  
Free Cell ◽  
Living Cells ◽  
Label Free ◽  
Controlled System ◽  
Chip Technology ◽  
Cell Monitoring ◽  
A Cell ◽  
On Chip

Label-free monitoring of living cells is used in various applications such as drug development, toxicology, regenerative medicine or environmental monitoring. The most prominent methods for monitoring the extracellular acidification, oxygen consumption, electrophysiological activity and morphological changes of living cells are described. Furthermore, the intelligent mobile lab (IMOLA) – a computer controlled system integrating cell monitoring and automated cell cultivation – is described as an example of a cell-based system for microphysiometry. Results from experiments in the field of environmental monitoring using algae are presented. An outlook toward the development of an organ-on-chip technology is given.

The nuclear pore complex, nuclear transport, and apoptosisThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 279-286 ◽  
Author(s):  
Birthe Fahrenkrog
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Morphological Changes ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Simple Fact ◽  
Cell Death Program ◽  
A Cell ◽  
Cellular Compartments ◽  
Selection Of

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.

How penicillin kills bacteria: progress and problems

1971 ◽  
Vol 179 (1057) ◽  
pp. 369-383 ◽  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bacterial Cell ◽  
Crosslinking Reaction ◽  
Free System ◽  
Whole Cells ◽  
Uridine Nucleotide ◽  
History Of ◽  
A Cell ◽  
Specific Inhibitors

Penicillins and cephalosporins are specific inhibitors of the biosynthesis of bacterial cell walls. This discovery was first made in 1957 and was based on two observations. First, penicillins induced the formation of protoplasts or spheroplasts in bacteria (organisms in which the cell wall has been lost or weakened) (Lederberg 1957). Secondly, a uridine nucleotide accumulated in Staphylococcus aureus and other bacteria inhibited by penicillin which had a striking relationship to the composition of the cell wall (Park & Strominger 1957). It was therefore suggested that this nucleotide was an activated precursor of the wall. Over the next decade, a great deal of work was carried out in order to elucidate the structure of the bacterial cell wall and the mechanism of its biosynthesis from the uridine nucleotides and other precursors (reviewed by Strominger 1970; Strominger & Ghuysen 1967; Ghuysen 1968). It was demonstrated that interpeptide cross-links were an important structural feature of the wall. Several kinds of experiments carried out with whole cells indicated that the final step in cell wall synthesis, the crosslinking reaction catalysed by a transpeptidase, was the site of action of penicillin (Wise & Park 1965; Tipper & Strominger 1965 a , b , 1968). Finally, in 1966, the transpeptidase catalysing this cross-linking reaction was obtained in a cell-free system and shown to be a penicillin-sensitive enzyme (Izaki, Matsuhashi & Strominger 1966, 1968). The history of these developments has been reviewed elsewhere (Strominger 1970), and in the present paper, attention will be focused on recent studies of the penicillin-sensitive transpeptidase and other penicillinsensitive activities found in bacterial cell membranes. First, however, it is necessary to describe briefly the structure of the cell wall of bacteria and the nature of the inhibited reactions. The walls of bacteria consist of glycan strands in which two sugars, acetylglucosamine (X) and acetylmuramic acid (Y), strictly alternate (figure 1). Four such glycan strands are represented in figure 1. The acetylmuramic acid residues of the polymer are substituted by a tetrapeptide (represented in the figure by open circles). The peptidoglycan strand (i.e., the glycan substituted by the tetrapeptide) are cross-linked to one another by means of an interpeptide bridge which is to some extent a genus-specific character­istic. In the genus Staphylococcus aureus , the interpeptide bridge is a pentaglycine chain (represented in figure 1 by the closed circles) which extends from the carboxyl group on the terminal D-alanine residue of the tetrapeptide to the ∊-amino group of lysine, the third amino acid in the tetrapeptide chain. The wall of S . aureus is a very tightly knit structure in that virtually every peptide subunit is cross-linked to another subunit by means of this interpeptide bridge. Penicillins and cephalosporins are specific inhibitors of the reaction in which the cross-link is actually formed. This step is the last reaction in wall synthesis.

scholarly journals The introduction of the C-22–C-23 ethylenic linkage in ergosterol biosynthesis

1968 ◽  
Vol 106 (3) ◽  
pp. 623-626 ◽  
Author(s):  
M Akhtar ◽  
M. A. Parvez ◽  
P. F. Hunt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Good Yield ◽  
The Other ◽  
Side Chain ◽  
Ergosterol Biosynthesis ◽  
Hydrogen Atoms ◽  
Whole Cells ◽  
C 23 ◽  
Methylene Derivative

Methods for the chemical synthesis of [23−3H2]lanosterol, [23,25−3H3]24-methyldihydrolanosterol and [24,28−3H2]24-methyldihydrolanosterol are described. It is shown that, in the biosynthesis of ergosterol from [26,27−14C2,23−3H2]lanosterol by the whole cells of Saccharomyces cerevisiae, one of the original C-23 hydrogen atoms is lost and the other is retained at C-23 of ergosterol. It is also shown that 24-methyldihydrolanosterol is converted into ergosterol in good yield and without prior conversion into a 24-methylene derivative. On the basis of these results possible pathways for the formation of the ergosterol side chain from a 24-methylene side chain are discussed.

Proliferation and Migration of Human Osteoblasts on Porous Three Dimensional Scaffolds

2010 ◽  
Vol 638-642 ◽  
pp. 506-511 ◽  
Author(s):  
Claudia Bergemann ◽  
Ernst Dieter Klinkenberg ◽  
Frank Lüthen ◽  
Arne Weidmann ◽  
Regina Lange ◽  
...  
Keyword(s):  
Cell Culture ◽  
Morphological Changes ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Porous Tantalum ◽  
Human Osteoblasts ◽  
A Cell ◽  
And Migration ◽  
Migration Of Cells

Porous tantalum (Ta) biomaterial is designed to function as a scaffold for osseous ingrowths and has found applications in orthopedics. Integration of this Ta foam into the neighboring bone requires that osteoprogenitor cells attach to the implant, grow into the scaffold, proliferate and differentiate to osteoblasts. The aim of the present study was to create an in vitro 3D model system to investigate the interaction of human osteoblasts with porous Ta in the depth of the corpus. To explore active migration of osteoblasts into the Ta scaffold two porous Ta discs (Zimmer, Poland) were horizontally fixed within a clamping ring. Thereby a 3D Ta module with 4 levels is generated, which is placed into a cell culture well with the appropriate medium. Osteoblast-like cells were seeded apical onto the Ta module and cultured for 7 days in humidified atmosphere. Active migration of cells into the scaffold was monitored by field emission scanning electron microscopy (FESEM) imaging of the apical, medial and basal layers. A problem in 3D cell culture is the nutrition of cells inside of the scaffold. Therefore morphological changes and differentiation of the cells in distinct layers were analyzed.

scholarly journals The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

1972 ◽  
Vol 127 (5) ◽  
pp. 833-844 ◽  
Author(s):  
C. G. Orpin ◽  
M. Knight ◽  
W. C. Evans
Keyword(s):  
Enzyme System ◽  
Soluble Enzyme ◽  
Bacterial Oxidation ◽  
Gram Negative ◽  
Whole Cells ◽  
Photodegradation Product ◽  
Enzyme Systems ◽  
A Cell ◽  
Methyl Derivatives ◽  
Bacterium Strain

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1′-dimethyl-4,4′-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [14C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[14C]methylisonicotinate, and no [14C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-14C]isonicotinate released 95% of the radioactivity as 14CO2. The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO2 by enzyme systems requiring NAD+.

scholarly journals Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence

1999 ◽  
Vol 67 (8) ◽  
pp. 3830-3835 ◽  
Author(s):  
J. R. McQuiston ◽  
R. Vemulapalli ◽  
T. J. Inzana ◽  
G. G. Schurig ◽  
N. Sriranganathan ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Genomic Dna ◽  
Homology Search ◽  
Side Chain ◽  
Broad Host Range ◽  
Genomic Fragment ◽  
Wild Type ◽  
Reading Frame ◽  
Whole Cells ◽  
Chain Synthesis

ABSTRACT We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kbXbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kbXbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been namedwboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype.B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.

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