SM Production Research Articles

Alterations in the silymarin metabolism in transgenic Silybum marianum cultured cells by the heterologous expression of the Arabidopsis thaliana V-myb myeloblastosis viral oncogene homolog transcription factor MYB12 and Cicer arietinum chalcone synthase

Silymarin (Sm), the main bioactive principle of Silybum marianum (L.) Gaernt. fruits, has strong antihepatotoxic properties and also exhibits antitumor and antiviral activity. This flavonolignan complex is synthesized from the flavonoid precursor taxifolin, which undergoes an oxidative coupling with the monolignol coniferyl alcohol. Attempts to produce Sm in plant cell suspensions have met with limited success, probably due to insufficient flux into the flavonoid branch pathway of Sm biosynthesis.In this study, it was attempted to increase Sm production in in vitro cultures of S. marianum by genetically engineering the flavonoid pathway. Using Agrobacterium tumefaciens strain C58C1, S. marianum cell suspension cultures were transformed either with the Arabidopsis thaliana transcription factor AtMYB12 (V-myb myeloblastosis viral oncogene homolog) (MYB) or with a chalcone synthase (CHS) gene from Cicer arietinum. A double transformant was also generated by introducing the CaCHS gene in AtMYB12-expressing cultures.The ectopic expression of AtMYB12 in cultures activated some of the genes involved in the flavonoid pathway, notably chalcone isomerase (CHI) and flavonoid 3′-hydroxylase (F3’H), whereas the secondary metabolites altered by the inserted gene were chlorogenic acid glycoside derivatives. Expression of CaCHS in the cultures led to the production of the flavanone naringenin (Ng), which accumulated as glycosides. The simultaneous expression of AtMYB12-CaCHS resulted in the production of lower amounts of chlorogenic derivatives but similar levels of Ng glycosides compared with the CaCHS lines. Both CaCHS lines and the double transformant could be maintained over prolonged subcultures without Ng production losses, confirming long-term and stable CaCHS transgene expression.In no case did the inserted genes have a discernable effect on Sm production compared with the untransformed cultures, neither under control culture conditions or in cultures elicited with MeJA.

Biotic and abiotic stress roles in drugs production through in vitro approaches in plants – a review

Abstract. Plant metabolic engineering is a modern discipline that promises to create opportunities in pharmaceutical industries to produce and biomedicine. Over the long period natural and synthetic plant hormones have had tremendous implications in callus/cell culture /suspension/ for secondary metabolites production (SMs). Generally, SMs plays a vital fundamental role in protecting the plant from biotic and abiotic attacks to which it may be subjected. This review article focused on the relationship between various factors related to the drug production. In medicinal plants, in vitro studies, based on biotic factors such as fungal/endo-phytic fungal elicitors/microbe-derived exogenous elicitor yeast extract (YE) were cross checked with the abiotic six factor groups, including auxins and cytokinins, gamma radiation, lights, temperature, carbon sources, photoperiods, precursor chemicals and plant metabolic enzymes. Moreover, key enzymes and gene networks can serve as a resource to selected potential targets for specific SMs production. This is the first review to describe the light factors needed for the SM production, which has favorable role for SMs. We envisage that the researcher can design how to modulate the stress factors in terms of drug improvement from medicinal plants.

Epigenetic modification enhances ergot alkaloid production of Claviceps purpurea.

To enhance ergot alkaloid production of Claviceps purpurea Cp-1 strain by epigenetic modification approach. The chemical epigenetic modifiers were screened to promote ergot alkaloid production of the Cp-1 strain. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was found to significantly enhance the alkaloid productivity of the strain. Particularly, the titers of total ergot alkaloids were gradually increased with the increase of SAHA concentration in the fermentation medium, and the highest production of ergot alkaloids could be achieved at the concentration of 500μM SAHA. Specially, the titers of ergometrine and total ergot alkaloids were as high as 95.4mg/L and 179.7mg/L, respectively, which were twice of those of the control. Furthermore, the mRNA expression levels of the most functional genes in the ergot alkaloid synthesis (EAS) gene cluster were up-regulated under SAHA treatment. It was proposed that SAHA might increase histone acetylation in the EAS gene cluster region in the chromosome, which would loosen the chromosome structure, and subsequently up-regulate the mRNA expression levels of genes involved in the biosynthesis of ergot alkaloids, thereby resulting in the markedly increase in the production of ergot alkaloids. The ergot alkaloid production by the C. purpurea Cp-1 strain can be effectively increased by the application of histone deacetylase inhibitor. Our work provides a reference for using the chemical epigenetic modifiers to improve SM production in other fungi.

Precise test of Higgs boson properties via triple Higgs boson production in vector boson fusion at future colliders

For certain classes of Beyond the Standard Model theories, including composite Higgs models, the coupling of the Higgs to gauge bosons can be different from the Standard Model one. In this case, the multi-boson production via vector boson fusion (VBF) can be hugely enhanced in comparison to the SM production one due to the lack of cancellation in longitudinal vector boson scattering. Among these processes, triple Higgs boson production in VBF plays a special role - its enhancement is especially spectacular due to the absence of background from transversely polarised vector bosons in the final state. While the rates from $pp\to jjhhh$ production in vector boson fusion are too low at the LHC and even at future 33 TeV $pp$ colliders, we have found that the 100 TeV $pp$ future circular collider (FCC) has the unique opportunity to probe the $hVV$ coupling far beyond the LHC sensitivity. We have evaluated the $pp\to jjhhh$ rates as a function of deviation from the $hVV$ coupling and have found that the background is much smaller than the signal for observable signal rates. We also found that the 100 TeV $pp$ FCC can probe the $hVV$ coupling up to the permille level, which is far beyond the LHC reach. These results highlight a special role of the $hhh$ VBF production and stress once more the importance of the 100 TeV $pp$ FCC.

Aspergillusfumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A.fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A.fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A.fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A.fumigatus.

Drell-Yan process as an avenue to test a noncommutative standard model at the Large Hadron Collider

We study the Drell-Yan process at the Large Hadron Collider in presence of the noncommutative extension of standard model. Using the Seiberg-Witten map, we calculate the production cross-section to the first order in the noncommutative parameter $\Theta_{\mu\nu}$. Although this idea is evolving for long, limited amount of phenomenological analysis was done so far and dominantly in the context of linear collider. Some outstanding feature from this non-minimal noncommutative standard model not only modify the couplings over SM production channel, also allow additional nonstandard vertices which can play a significant role. Hence in the Drell-Yan process, as studied in present analysis, one also needs to account for gluon fusion process at the tree level. Some of the characteristic signatures such as oscillatory azimuthal distributions are outcome of the momentum dependent effective couplings. We explore the noncommutative scale $\Lambda_{NC} \ge 0.4~\rm{TeV}$ considering different machine energy ranging from $7~\rm{TeV}$ to $13~\rm{TeV}$.

Gene expression and flavonolignan production in fruits and cell cultures of Silybum marianum

The hepatoprotectant flavonolignan silymarin (Sm) is synthesized through 4-coumaroyl-CoA, which enters both the flavonoid and the monolignol pathway giving the two immediate precursors taxifolin (Tx) and coniferyl alcohol (CA), respectively. Sm formation occurs via oxidative radicalization of Tx and CA and is accumulated at high levels at final stages of maturation of Silybum marianum fruits. By contrast, Sm production is severely reduced in cell cultures of this species, although suspensions are able to excrete Sm compounds into the medium upon elicitation with methyl jasmonate (MeJA) or cyclodextrins (CD). Knowledge of gene expression is important to understand Sm dynamics and to develop strategies aimed at increasing production by means of cell cultures but, to date, only one gene of the pathway (chalcone synthase, SmCHS) has been cloned. Therefore, to elucidate the relationship between expression of Sm pathway genes and production of these metabolites, four cDNA fragments of genes putatively involved in flavonolignan biosynthesis, chalcone isomerase, flavanone 3-hydroxylase, flavonol 3′-hydroxylase and cinnamyl alcohol dehydrogenase, were isolated from Sm producing S. marianum fruits and their expression, together with that of the SmCHS, were studied both in fruits at different maturation stages and in elicited cell suspensions. Combined results at both transcript expression and metabolite levels at three different stages of fruit maturation revealed that the formation of the flavonoid moiety precedes flavonolignan biosynthesis, being Sm accumulation associated to expression of the monolignol pathway. There was not detectable accumulation of transcripts in cell suspensions, however, elicitation with MeJA or CD notably induced expression of the studied fragments. These results indicate that the five genes expressed during maturation of S. marianum fruits may contribute to observed increases in flavonolignan accumulation upon treatment of cell cultures with elicitors.

Comparison of periodic and aperiodic supermirrors

The recipe used nowadays in the neutron supermirror production gives aperiodic multilayers, i.e. the layer thickness is changing from bilayer to bilayer. An algorithm recently proposed by V. Ignatovich and Carron gives supermirrors composed of periodic stacks of bilayers. Samples were prepared according to both methods and their measured reflectivity is compared. The effect of different possible faults in manufacturing is investigated by numerical calculations and a quantitative comparison is given for the different supermirror recipes. The results confirm the applicability of both design methods for practical SM production.

Discriminating Higgs production mechanisms using jet energy profiles

We present a new tool for precision measurements of the Higgs boson production mechanisms at the LHC. We study events with a Higgs boson produced with two forward jets. Even with fairly stringent cuts, one expects a significant contamination of gluon fusion (GF) in addition to vector-boson fusion (VBF) in the event sample. By measuring the jet energy profile of the most central jet, we find that SM production can be distinguished from either pure VBF or pure GF at the $5\sigma$ level with 100 fb$^{-1}$ of luminosity at the 14 TeV LHC. Moreover, this discrimination technique can be used to validate or rule out new physics models that predict similar observable branching fractions as the 125 GeV SM Higgs but have different production mechanisms.

Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.

Light stops from Seiberg duality

If low-energy supersymmetry is realized in nature, a seemingly contrived hierarchy in the squark mass spectrum appears to be required. We show that composite supersymmetric theories at the bottom of the conformal window can automatically yield the spectrum that is suggested by experimental data and naturalness. With a non-tuned choice of parameters, the only superpartners below one TeV will be the partners of the Higgs, the electroweak gauge bosons, the left-handed top and bottom, and the right-handed top, which are precisely the particles needed to make weak scale supersymmetry breaking natural. In the model considered here, these correspond to composite (or partially composite) degrees of freedom via Seiberg duality, while the other MSSM fields, with their heavier superpartners, are elementary. The key observation is that at or near the edge of the conformal window, soft supersymmetry breaking scalar and gaugino masses are transmitted only to fundamental particles at leading order. With the potential that arises from the duality, a Higgs with a 125 GeV mass, with nearly SM production rates, is naturally accommodated without tuning. The lightest ordinary superpartner is either the lightest stop or the lightest neutralino. If it is the stop, it is natural for it to be almost degenerate with the top, in which case it decays to top by emitting a very soft gravitino, making it quite difficult to find this mode at the LHC and more challenging to find SUSY in general, yielding a simple realization of the stealth supersymmetry idea. We analyze four benchmark spectra in detail.

Methyl jasmonate increases silymarin production in Silybum marianum (L.) Gaernt cell cultures treated with β-cyclodextrins

Silymarin (Sm) from the fruit of Silybum marianum is an isomeric mixture of pharmacologically active flavonolignans which are formed by oxidative coupling of taxifolin (Tx) and coniferyl alcohol (CA). Suspension cultures of this plant constitutively secrete small amounts of Sm into the extracellular medium. Production can be increased by inclusion of cyclodextrins (CDs) in cultures. Both hydroxylated (RHCD) and dimethylated (RMCD) CDs strongly induced prompt accumulation of CA in the medium followed by a late production of flavonolignans. Simultaneous addition of methyl jasmonate (MJ) and RMCD to cells did not significantly modify CA release or flavonolignan accumulation. Delayed addition of MJ to cultures subcultivated in medium containing RMCD markedly influenced Sm production by promoting conversion of the previously formed CA precursor.

Are polyamines directly involved in silymarin production in the milk thistle [Silybum marianum (L.) Gaernt

The possible role of polyamines (PAs) in the regulation of silymarin (Sm) production in milk thistle [Silybum marianum (L.) Gaernt] cell suspension cultures was studied in a young cell culture line (H2 line) and in a synchronized cell line (>3 years; H1 line). The effect of two exogenous PAs, putrescine (Put) and spermidine (Spd), and a number of metabolic inhibitors (L-canavanine, DL-α-difluoromethylornithine, methylglyoxal-bis-guanylhydrazone, cyclohexylamine) on the production of Sm during the growth cycle were analyzed. The results suggest that PAs are not directly involved in the Sm synthesis pathway. In our cell culture system, Sm production and PA contents were determined by the age of the suspension culture cells: with increasing age, the suspension culture cells showed a decreasing capacity to reach the stationary phase during prolonged subculture that was associated with a decreased production of Sm, a steady increase in PA content, and a constant Put/Spd ratio. The synchronization of dividing cells from the S. marianum H1 line did not modify this behaviour. In young cell suspensions, maximum Sm production occurred in the stationary phase, concurrent with the cellular PA contents reaching their minimum value. At the start of the stationary phase, the high percentage of cells in the growth phases (G0/G1) and a transient increase in the Put/Spd ratio were accompanied by maximum Sm production and a blockade of cell division.

SM and MSSM Higgs boson production: spectra at large transverse momentum

Strategies for Higgs boson searches require the knowledge of the total production cross section and the transverse momentum spectrum. The large transverse momentum spectrum of the Higgs boson produced in gluon fusion can be quite different in the Standard Model and the Minimal Supersymmetric Standard Model. In this paper we present a comparison of the Higgs transverse momentum spectrum obtained using the PYTHIA event generator and the HIGLU program as well as the program HQT, which includes NLO corrections and a soft gluon resummation for the region of small transverse momenta. While the shapes of the spectra are similar for the Standard Model, significant differences are observed in the spectra of Minimal Supersymmetric Standard Model benchmark scenarios with large tan(beta).

Low scale gravity and extra dimensions at HERA, LEP and Tevatron

Publisher Summary This chapter provides an overview of low scale gravity and extra dimensions (EDs) at HERA, LEP, and Tevatron. At HERA, the H1 and ZEUS experiments have searched for ED using the complete statistics corresponding to 16 (112) pb-1 in e− p (e+ p) scattering. The interference of the G exchange with the SM ϒ and Z processes leads to an effective contact type interaction with a coupling G=λ/μS4, where the parameter λ depends on the details of the theory and is expected to be of order unity. At hadronic colliders, the virtual effects of the graviton in dilepton production in the s channel can come not only from q q ¯ annihilation but also from gluon fusion. For diphoton production, there is interference between the t channel SM production and the s channel production mediated by a graviton. At LEP, the most favorable process is e+e− → γG. The differential cross-section for this process depends on both the gravity scale MD and the number n of ED. In presence of ED, the distributions of the missing mass and polar angle θ would be enhanced at high missing mass and large cosθ. At present, the limits obtained at the three highest energy colliders are comparable of the order of 1 TeV and the searches have been made in a large variety of channels reinforcing the confidence in this limit. In future, this domain will be explored at the Tevatron with sensitivity up to about 2 TeV with full luminosity.

Breakthrough of SM production technology by ECUST

Breakthrough of SM production technology by ECUST

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2)

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network

Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system

A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression. The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis. A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription. The StrR product, in turn, activates the other Sm-biosynthesis genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene. Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes. It is most likely that AfsR and AfsK compose a two-component regulatory system. Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1. In addition to the afsR system, another pair of closely located genes that encode proteins with great similarities to the typical proteins of the prokaryotic two-component regulatory system was cloned by utilizing their abilities to confer pigment production on Streptomyces lividans.

Streptomycin biosynthesis and its regulation in Streptomycetes

New insights into the gene orders, structures, evolution, and functions of streptomycin (Sm) biosynthetic genes ( str) were gained via hybridization studies, determination of nucleotide sequences, and measurement of expression in the str gene clusters of Streptomyces griseus and S. glaucescens. Both str clusters showed considerable divergence in macro and micro structure. Genes putatively involved in pathways leading to the (dihydro-)streptose and N- methyl- L-glucosamine moieties of Sm were identified. Additional regulatory elements, such as gene strS and conserved TTA codons in the N-terminal sections of reading frames, are reported. Evidences for the involvement of physiological state, signal transduction, and activators in the control of Sm production are presented.

Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone controlling streptomycin (Sm) production, Sm resistance and sporulation in Streptomyces griseus. In order to identify A-factor-dependent promoters in the Sm biosynthetic gene cluster, a new promoter-probe plasmid with a low copy number was constructed by using an extremely thermostable malate dehydrogenase gene as the reporter. Of the three promoters in the Sm production region that includes strR, aphD and strB, only the promoter of strR, which codes for a putative regulatory protein, was found to be directly controlled by A-factor. This was also confirmed by S1 nuclease mapping. The region essential for its A-factor-dependence was determined to be located 430-330 base pairs upstream of the transcriptional start point. Increase in the copy number of the strR promoter region did not lead to a corresponding increase in the total promoter activity, probably due to titration of a putative activator which binds to the enhancer-like region and controls the expression of the strR promoter. This putative activator is apparently distinct from the A-factor-receptor protein. The aphD gene, which encodes the major Sm resistance determinant, Sm-6-phosphotransferase, was transcribed mainly by read-through from the A-factor-dependent strR promoter; this accounts for the prompt induction of Sm resistance by A-factor.