Showing papers on "Lanosterol published in 2004"

Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase

Abstract: In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.

Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans

Abstract: Objectives: The aim of this study was to identify changes in the gene expression profile of Candida albicans associated with the acquisition of experimentally induced resistance to amphotericin B and fluconazole. Methods: C. albicans SC5314 was passed in increasing concentrations of amphotericin B to generate isolate SC5314-AR. Susceptibility testing by Etest revealed SC5314-AR to be highly resistant to both amphotericin B and fluconazole. The gene expression profile of SC5314-AR was compared with that of SC5314 using DNA microarray analysis. Sterol composition was determined for both strains. Results: Upon examination of MICs of antifungal compounds, it was found that SC5314-AR was resistant to both amphotericin B and fluconazole. By microarray analysis a total of 134 genes were found to be differentially expressed, that is up-regulated or down-regulated by at least 50%, in SC5314-AR. In addition to the cell stress genes DDR48 and RTA2, the ergosterol biosynthesis genes ERG5, ERG6 and ERG25 were up-regulated. Several histone genes, protein synthesis genes and energy generation genes were down-regulated. Sterol analysis revealed the prevalence of sterol intermediates eburicol and lanosterol in SC5314-AR, whereas ergosterol was the predominant sterol in SC5314. Conclusion: Along with changes in expression of these ergosterol biosynthesis genes was the accumulation of sterol intermediates in the resistant strain, which would account for the decreased affinity of amphotericin B for membrane sterols and a decreased requirement for lanosterol demethylase activity in membrane sterol production. Furthermore, other genes are implicated as having a potential role in the polyene and azole antifungal resistant phenotype.

CYP51 from Trypanosoma brucei is obtusifoliol-specific.

Abstract: New isoforms of CYP51 (sterol 14alpha-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei(TB), T. vivax, T. cruzi, and Leishmania major. The sequences share approximately 80% amino acid identity and are approximately 25% identical to sterol 14alpha-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT)) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min(-1)). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14alpha-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.

The Candida albicans lanosterol 14-α-demethylase (ERG11) gene promoter is maximally induced after prolonged growth with antifungal drugs

Abstract: The azole antifungal drugs that target lanosterol 14-α-demethylase, encoded by the ERG11 gene, are used to treat a variety of infections caused by Candida albicans. Azoles are known to induce expression of ERG11 mRNA. The ERG11 promoter was cloned 5′ of the luciferase-coding region, and the induction of ERG11 expression by azoles was monitored by luciferase assays. Maximal induction of the ERG11 promoter by azoles occurs not during logarithmic growth but after the diauxic shift and requires azoles to be present throughout logarithmic growth. The effects of pH, carbon source, and aerobic or anaerobic growth on induction of the ERG11 promoter by azoles were analyzed. Treatment with terbinafine and fenpropimorph, which target other enzymes in the ergosterol biosynthetic pathway, also resulted in a delayed induction of ERG11 promoter activity. Nascent sterol synthesis was shown to parallel ERG11 promoter activity, and total sterols were reduced coincident with the timing of ERG11 promoter activation. These results as a whole suggest that expression of the ERG11 promoter is regulated in response to sterol depletion.

Homology modeling of lanosterol 14α-demethylase of Candida albicans and Aspergillus fumigatus and insights into the enzyme-substrate interactions

Abstract: The crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis(MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14α-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14α-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P45014DM. The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues...

New aspects on lanosterol 14α-demethylase and cytochrome P450 evolution: Lanosterol/cycloartenol diversification and lateral transfer

Abstract: Sterol 14α-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups. CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms. We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily. Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches. Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs. Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch. A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability). This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis. The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D. discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data. Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes. Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily. Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin.

Lanosterol and Cholesterol-Induced Variations in Bilayer Elasticity Probed by 2H NMR Relaxation

Abstract: The influences of lanosterol on lipid bilayers have been compared to those of cholesterol by combining deuterium (2H) NMR spin relaxation studies with segmental order parameter measurements. For bilayers of 1,2-diperdeuteriomyristoyl-sn-glycero-3-phosphocholine (DMPC-d54), the results are consistent with a square-law dependence of the 2H Zeeman relaxation rates (R1Z) on the corresponding order parameters (SCD). This behavior is indicative of relatively slow order fluctuations, for example, due to quasi-elastic bilayer disturbances. Significant differences are found in the influences of lanosterol versus cholesterol on the microscopic NMR observables; although lanosterol produces smaller order parameters than cholesterol, it leads to larger relaxation rates. By correlating the NMR relaxation behavior with the order parameters, the results are explained by a progressive reduction of the bilayer elasticity, which parallels the biosynthetic pathway from lanosterol to cholesterol.

A Functional Cytochrome P450 Lanosterol 14α-Demethylase CYP51 Enzyme in the Acrosome: Transport through the Golgi and Synthesis of Meiosis-Activating Sterols

Abstract: Mammalian lanosterol 14α-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fr...

Enzymatic Formation of Multiple Triterpenes by Mutation of Tyrosine 510 of the Oxidosqualene‐Lanosterol Cyclase from Saccharomyces cerevisiae

Abstract: Triterpene cyclases constitute a family of enzymes that catalyze diverse and complex carbocationic cyclization/rearrangement reactions of squalene and (3S)-2,3-oxidosqualene (OS) to generate a distinct array of sterols and triterpenes. A major determinant for the triterpenoid diversity is believed to be the precise control of conformation between substrate and enzyme, as well as the position of the carbocation intermediate formation. For example, both oxidosqualene-lanosterol cyclase (ERG7, EC 5.4.99.7) and oxidosqualene-cycloartenol synthase (CAS, EC 5.4.99.8) bind oxidosqualene in a chair–boat– chair conformation, initiate and propagate cyclization to form a protosteryl cation, and then promote 1,2-shifts of hydride and methyl groups to the lanosteryl C8 cation. The lanosterol formation is accomplished through the final deprotonation, abstracting a proton originally at C9 or after a hydride shift from C9 to C8. Cycloartenol is formed after a hydride shift from C9 to C8, followed by 9b,19-cyclopropane ring closure. Elegant molecular-genetic and bioorganic investigations have recently identified several amino acid residues that are critical in probing putative active sites and determining prod-

Oxysteroids: a new class of steroids with autocrine and paracrine functions

Abstract: Oxysteroids are a new classification for sterol intermediates in cholesterol synthesis that undergo enzyme-catalyzed stereo-specific 25R,26-hydroxylation and thus bypass cholesterol as the expected end-product. Recently, they were identified in micromolar amounts in the plasma of patients with Smith-Lemli-Opitz syndrome (SLOS). An additional three oxysteroids, the 25,26-hydroxy derivatives of lanosterol, zymosterol, and desmosterol, respectively, were generated in vitro by CYP27A1-transfected bacteria. As there are 19 steps between cholesterol and lanosterol, the first post-squalene sterol, a potentially large class of oxysteroids exists. Limited studies of 25r,26-7-dehydrocholesterol indicate a traditional role as a ligand for nuclear receptors, but complete evaluation of oxysteroids for novel biologic activities is lacking. Currently, the lack of authentic oxysteroid standards limits both their detection in biologic fluids and evaluation of their biologic effects.

Induction of CYP3A by 2,3-oxidosqualene:lanosterol cyclase inhibitors is mediated by an endogenous squalene metabolite in primary cultured rat hepatocytes.

Abstract: The effects of inhibitors of 2,3-oxidosqualene:lanosterol cyclase (cyclase) on cytochrome P450 expression were investigated in primary cultures of rat hepatocytes. Treatment of hepatocyte cultures for 24 h with either of the inhibitors [4'-(6-allyl-methyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071) or trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79) selectively increased CYP3A mRNA and immunoreactive protein contents, with maximal accumulations occurring at 3 x 10(-5) M Ro 48-8071 and 10(-4) M BIBX 79. The abilities of Ro 48-8071, BIBX 79, and 3beta-(2-diethylaminoethoxy)androst-5-en-17-one.HCl (U18666A) to induce murine CYP3A were abolished in hepatocyte cultures prepared from pregnane X receptor (PXR)-null mice, and cotransfection of primary cultured rat hepatocytes with a dominant-negative PXR prevented cyclase inhibitor-inducible luciferase expression from a PXR-responsive reporter plasmid. Cyclase inhibitor-mediated CYP3A mRNA induction was eliminated when primary cultured rat hepatocytes were cotreated with any of the following agents that inhibit steps upstream of cyclase in the cholesterol biosynthetic pathway: squalestatin 1 (squalene synthase inhibitor), (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598, squalene monooxygenase inhibitor), or pravastatin (HMG-CoA reductase inhibitor). Ro 48-8071-inducible CYP3A mRNA expression was restored when pravastatin-treated cultures were incubated with medium containing mevalonate. The concentration-dependence of Ro 48-8071-mediated CYP3A mRNA induction corresponded to the cellular contents of metabolically labeled squalene 2,3-oxide and squalene 2,3:22,23-dioxide, but not 24(S),25-epoxycholesterol. These results indicate that cyclase inhibitors are capable of inducing CYP3A expression in primary cultured rat and mouse hepatocytes and that the effect is mediated as a consequence of cyclase blockade through the evoked accumulation of one or more squalene metabolites that activate the PXR.

Isolation, characterization, and regulation of the Candida albicans ERG27 gene encoding the sterol 3-keto reductase.

Abstract: The Candida albicans ERG27 gene which encodes the 3-keto reductase enzyme required for sterol C-4 demethylation was isolated and found to encode a 349 amino acid protein that is 60% identical at the amino acid level to the Saccharomyces cerevisiae Erg27p. A C. albicans erg27 null was created in a strain containing an integrated ERG27 rescue cassette under the control of the pMAL2 inducible promoter. The C. albicans erg27 strain was able to grow only in the presence of maltose indicating that the ERG27 gene is essential. The C. albicans erg27 null showed complete loss of both 3-keto reductase and oxidosqualene cyclase (Erg7p) activities compromising all sterol synthesis. These results suggest that Erg27p inhibitors might be effective antifungals. To explore ERG27 regulation, an erg11 null strain was generated. C. albicans erg6 and erg24 mutants were also employed along with the inhibitors, itraconazole and zaragozic acid A, to characterize ERG27 expression using Northern analysis. Expression was increased two- to fourfold in erg11, erg6 and erg24 backgrounds. However, itraconazole which targets Erg11p (lanosterol demethylase) increased ERG27 expression 10-fold and zaragozic acid A which targets the Erg9p (squalene synthase) increased ERG27 expression fivefold. The azole and erg11 results support other observations that azoles may affect non-sterol targets.

Pyridines and pyrimidines mediating activity against an efflux-negative strain of Candida albicans through putative inhibition of lanosterol demethylase

Abstract: The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific β-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 μg · ml−1). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 μg · ml−1), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

Design,Synthesis and Antifungal Activity of Novel Triazole Derivatives

Abstract: Twenty-one 1-(1H-1,2,4-triazolyl)-2-(2,4-diflurophenyl)-3-(4-substituted-1-piperazinyl)-2-propanol derivatives were designed and synthesized,on the basis of the active site of lanosterol 14α-demethylase.In vitro antifungal activities showed that some of the target compounds had higher antifungal activity and broader antifungal spectrum than fluconazole.

Disruption of the Candida albicans CYB5 Gene Results in Increased Azole Sensitivity

Abstract: The fungal end product sterol, ergosterol, and its biosynthetic pathway are the targets of the majority of the antifungal compounds currently in use for human infections and agricultural applications. The primary class of compounds used in human infections is the azoles, drugs that inhibit the C-14 demethylation of the pathway intermediate lanosterol. Overuse of the azoles, especially in immunocompromised patients, has led to increases in the incidence of antifungal resistance (5). As resistance levels to these compounds rise, the need to identify new targets for antifungal therapy expands. Several reactions in the ergosterol biosynthetic pathway of the pathogenic fungus Candida albicans have been identified (1, 18, 26) as required for viability and, thus, might be subject to exploration as new antifungal sites. Inhibition of these steps could prove to be effective antifungal interventions because this organism cannot import exogenous sterol (19, 29), making sterol biosynthesis obligatory. Sterol biosynthesis in fungi is an exclusively aerobic process because several steps in the ergosterol biosynthetic pathway require molecular oxygen or heme, which are also synthesized only under aerobic conditions and used primarily in cytochrome-mediated reactions. Among the latter are the cytochrome P-450-dependent step in lanosterol demethylation (encoded by CYP51, also referred to as ERG11 and ERG16), the cytochrome P-450-dependent desaturation step at C-22 (encoded by CYP61, also referred to as ERG5), and the cytochrome b5 requiring C-5 desaturation (encoded by ERG3). In the case of the cytochrome P-450 steps, two distinct enzymes are involved (11, 12), but each requires the same electron donor, the NADPH cytochrome P-450 oxidoreductase (encoded by NCP1). Ncp1p also donates electrons in the presterol, oxygen-requiring reaction catalyzed by squalene epoxidase (32). The cytochrome b5 step in the pathway utilizes NADH-cytochrome b5 reductase or NADPH-cytochrome c reductase as electron carriers in the creation of the double bond at C5-6 (25). Sutter and Loper (27) reported that disruption of the Saccharomyces cerevisiae NCP1 gene is not lethal despite the fact that two steps in the pathway utilize this electron donor and that one of the steps, the C-14 demethylation of lanosterol, has proven to be essential based on the fact that disruption of the cytochrome P-450 demethylase gene (CYP51) results in nonviability (17). The presence of an alternative electron carrier, perhaps cytochrome b5, was postulated to explain the viability of the ncp1 mutant (13). This substitution of function by cytochrome b5 was confirmed in a follow-up study (20). Similarly, the gene encoding cytochrome b5 in S. cerevisiae was found to be nonessential (28). Disruption of CYB5 generates no growth phenotype in a wild-type background but results in lethality when present in a ncp1 background (28). This suggests that NCP1 might provide a reciprocal function for the missing cytochrome b5 protein. Ncp1p and Cyb5p appear to have overlapping functions in S. cerevisiae. One of the phenotypes of ncp1 is an increased sensitivity to azole antifungals (27). This is postulated to be due to the increased sensitivity of the substitute electron carrier in the C-14 demethylation reaction and suggests a mechanism of action for azoles beyond interacting with Cyp51p. A more recent study (28) has confirmed the azole sensitivity of the ncp1 phenotype and has demonstrated that ncp1 cells still produce about 25% of the ergosterol produced by NCP1 strains. In addition, elevated levels of lanosterol were not detected, indicating that the cells were able to complete the C-14 demethylation reaction as well as the other reactions where Ncp1p normally functions. Based on an in vitro assay (13) and on suppression of the ncp1 phenotype by CYB5 (28), Cyb5p could be the replacement electron donor. Based on the increased azole sensitivity of ncp1 strains coupled with the fact that required reactions in sterol biosynthesis in S. cerevisiae and C. albicans can differ (16), it is worthwhile to investigate the characteristics of the cyb5 and the ncp1 phenotypes in this pathogenic fungus. This report investigated whether CYB5 is an essential gene and whether Cyb5p may be a potential drug target. This report also employed various methods of gene disruption due to difficulties in isolating homozygous mutants for the determination of gene essentiality.

Density functional study on formation of A and B rings in conversion of 2,3-oxidosqualene to lanosterol

Abstract: A density functional computational study has been performed on a model system for the formation of the A and B rings in the oxidosqulene cyclase pathway for the cyclization of squalene to lanosterol. Unlike what had previously been found for the squalene hopene cyclase pathway, the formation of the A and B rings was found not to be concerted. Two intermediates were located on the reaction pathway during the formation of the B ring, though they were found to be very shallow minima on a quite flat part of the potential surface. It is unlikely that in the enzymatic pathway these intermediates would play an important role, if indeed they exist at all.

Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae.

Abstract: Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1–1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.

Chemical Constituents of Stationary Cultured Mycelia of Inonotus obliquus

Abstract: Seven compounds were isolated from stationary cultured mycelia of Inonotus obliquus, of which several of them showed certain antitumor activities. Seven compounds were identified as lanosterol(l), inotodiol(2), trametenolic acid(3), 3 ,22,25-trihydroxy-lanosta-8-ene(6), 3 ,22-dihydroxy-lanosta-8,24-diene(A), 3 -hydroxylanosta-8,24-dien-21-al(B) and methyl trametenolate(C), respectively. The precursor compound of sclerotium obtained from shaking-cultured and stationary-cultured mycelia of Inonotus obliquus is lanosterol. Ergosterol and ergosterol peroxide were obtained by shaking-culture, and the substituted compounds of C-21 and C-22 of lanosterol were obtained by stationary culture.