From the Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2UH, the § Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, and the ¶ Neonatal Screening Laboratory, Sheffield's Children's Hospital, Western Bank, Sheffield S10 2UH, United Kingdom
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ABSTRACT |
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The disruption of Saccharomyces cerevisiae NADPH- cytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.
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INTRODUCTION |
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NADPH-cytochrome P450 oxidoreductase (CPR)1 (EC 1.6.2.4) is required for microsomal eukaryotic cytochrome P450 (CYP) monooxygenase activity, transferring either both electrons or (sometimes) the first electron for these reactions (1). The CYP enzymes are involved in the metabolism of foreign compounds, such as lipophilic pollutants, pesticides, and drugs, as well as in many biosynthetic reactions (for instance, in steroid, alkaloid, and terpenoid biosynthesis). Although in plants there is CPR diversity, in animal and fungal systems only one CPR has been identified; it functions with the many members of the microsomal P450 superfamily in a particular organism (2). The soluble domain of the enzyme can be generated for structural studies via x-ray crystallography (3, 4), but it was previously reported to be unable to support mammalian CYP activity where the N-terminal membrane anchor was required (5). We are interested in the yeast CPR, and we developed and characterized systems to examine the function of the soluble domain of the enzyme with comparison to human CPR. We show here, using whole transformants and cell-free systems, that the soluble yeast enzyme can support CYP activity, in contrast to human CPR, which is shown to be inactive.
Within the CYP superfamily, CYP51 is the only family found in animal, plant, and fungal kingdoms, and it represents an ancient metabolic role for CYP in sterol biosynthesis, undertaking C14 demethylation via three sequential hydroxylations (6). The fungal enzyme is the target for azole antifungals, which are selective in their inhibition over the human and plant orthologues, are central to antifungal chemotherapy, and represent about one-third of the agricultural fungicides used. Two other enzymes of fungal ergosterol biosynthesis require CPR, CYP61 (a sterol 22-desaturase (7-9)), and a non-CYP monooxygenase, squalene epoxidase (10). The former may also be present in plants, in which 22-desaturation is observed, unlike in animals, but the latter is present in all organisms producing sterols (11).
As expected for an antifungal target, gene disruption of CYP51 was observed to be lethal, but the strain could be rescued by providing an ergosterol supplement that could be taken up only anaerobically (12). In contrast, gene disruption of yeast CPR produced viable mutants (13) and ergosterol was present.2 No additional CPR genes could be detected or were subsequently revealed within the yeast genome. In reconstituted assays with purified CYP, enzyme cytochrome b5 can act as alternative donor for the second electron required for monooxygenase activity (14) and may have been supporting catalytic activity in the disruptant. Supporting this concept was the observation that the gene encoding cytochrome b5 can act as a suppressor of a CPR gene disruption phenotype, noted by Sutter and Loper (13), namely hypersensitivity to the CYP51 inhibitor ketoconazole (15). The hypersensitivity suggested that a large reduction in ergosterol biosynthesis might have occurred, making the strain more sensitive, with reduction in ergosterol levels below the critical level occurring at a lower concentration of drug.
Here, we present the first full biochemical characterization of a yeast strain disrupted in the gene encoding CPR, including measurement of activity, enzyme localization, and qualitative and quantitative investigation of sterol profiles. It is shown that an absence of yeast CPR does not result in a dramatic reduction in ergosterol synthesis in the cell. Furthermore, the ability of an N-terminal truncated soluble yeast enzyme to complement a strain containing no CPR and reconstitute CYP activity is demonstrated. This is in direct contrast to genetically engineered soluble human CPR, which is shown to be inactive, thus revealing a fundamental structural difference between the yeast and mammalian CPR forms.
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EXPERIMENTAL PROCEDURES |
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Strains--
Escherichia coli strain DH5 was used
for cloning, and Saccharomyces cerevisiae strain JL20
(MAT a, leu2-3, 2-112, his4-519, ade1-100,
ura3-52) was used for expression and gene disruption.
Construction of Expression Vectors--
The expression vectors
containing yeast and human CPR, and the 5'-end truncated
CPR forms were constructed in YEp51, a galactose inducible
yeast expression plasmid. Yeast CPR and CPR
(33) genes were isolated by PCR using pFBY4 (16) containing
yeast CPR as template. The 5'-sense oligonucleotide
primers were 5'-CCCGTCGACATCATGCCGTTTGGAATAGACAAC-3' for yeast
CPR and 5'-CCCGTCGACATCATGTCCGATGACGGAGATATC-3' for yeast CPR (
33) The latter corresponds to the sequence
starting from methionine codon of the 33 amino acid from the N terminus and contained a SalI site at the 5'-end. The 3'-antisense
oligonucleotide primers (5'-CCCAAGCTTTTACCAGACATCTTCTTGGTA-3' for yeast
CPR and CPR (
33)) encoded a HindIII
site at the 3'-end. The reaction conditions were as follows: 94 °C
(denaturation) for 1 min, 45 °C (annealing) for 1 min and 72 °C
(extension) for 5 min with 2 min ramp for first 5 cycles and 94 °C
for 1 min and 72 °C for 5 min for the remaining cycles in a 30-cycle
reaction. PCR was carried out using Pfu polymerase
(Stratagene) and a Perkin-Elmer DNA thermal cycler. Construction of
expression vectors for human CPR, a truncated human
CPR equivalent to yeast CPR (
33) (human
(
50)), and human CPR digested with trypsin (
55) were produced in
a similar way using human CPR as a template. The 5'-sense
oligonucleotide primers (each encoding a SalI restriction
site) were 5'-CCCGTCGACATCATGGGAGACTCCCACGTGGAC-3' for human
CPR, 5'-CCCGTCGACATCATGCCCGAGTTCACCAAAATTC-3' for
human CPR (
50), and
5'-CCCGTCGACATCATGATTCAGACATTGACCTCCT-3' for human CPR
(
55). The 3'-antisense oligonucleotide primer for human
CPR, CPR (
50), and CPR (
55),
encoding a HindIII site following the stop codon, was
5'-CCCAAGCTTCTAGCTCCACACGTCCAGGGA-3'. PCR conditions for
generation of the human CPR fragments were 1 min at 94 °C (denaturation), 2 min at 45 °C (annealing), and 1.5 min at 72 °C (extension) in a 30-cycle reaction using Pfu DNA polymerase.
The target fragments were gel purified, digested with SalI
and HindIII, and cloned into YEp51. Transformants were
screened by restriction digestion and confirmed by sequencing. All DNA
manipulations and transformations were done using standard protocols
(17).
CPR Gene Disruption in JL20-- In the JL20 strain, the chromosomal CPR gene was disrupted by inserting a URA3 to generate the JL20 (CPR::URA3) strain (13). The 0.7-kilobase BamHI internal fragment of the CPR gene in the CPR:YEp51 construct was replaced by the 1.1-kilobase URA3-containing HindIII fragment isolated from plasmid pJJ244 (18) by filling in and blunt-ended ligation to obtain a CPR::URA3:YEp51 construct. Chromosomal CPR in the haploid JL20 yeast strain was then inactivated by transplacement using the SalI-HindIII fragment containing CPR::URA3, and the disruption was confirmed by PCR, using the cells of URA3 colonies of the transformed JL20 strain, the primers and conditions mentioned above (the cells were heated in a microwave for 30 s and quickly cooled on ice for lysis before being subjected to PCR), and ketoconazole susceptibility tests.
Sterol Isolation and Analysis-- Yeast cells harvested from 100 ml of culture were resuspended in 3 ml of methanol, 2 ml of 60% (w/v) KOH in water, and 2 ml of 0.5% (w/v) pyrogallol dissolved in methanol and saponified by heating at 90 °C for 1 h. Nonsaponifiable lipids (sterols) were extracted from the saponified mixture three times with 5 ml of hexane each time, pooled, and dried under nitrogen. The sterols were suspended in 100 µl of toluene and heated at 60 °C for one hour for silylation after adding 20 µl of bis(trimethylsilyl) trifluoride. The silyl sterols were analyzed by gas chromatography/mass spectrometry (VG 12-250; VG BIOTECH) by using split injections with a split ratio of 20:1. Sterol identification was by reference to relative retention times and mass spectra reported previously (19, 20).
Immunoblot Analysis-- SDS-10% polyacrylamide gel electrophoresis, nitrocellulose filter transfer, and immunodetection of protein were performed as described previously (21, 22). Yeast CPR antiserum was kindly provided by J. C. Loper of Cincinnati University. Anti-CPR IgG was purified from the CPR antiserum as described (23).
Heterologous Expression in the JL20 Strain-- The JL20 transformants carrying native and truncated yeast and human CPR expression vectors were grown in yeast minimal medium containing Difco yeast nitrogen base without amino acids (1.34%, w/v), 20 µg/ml L-histidine, and glucose (2%, w/v) at 30 °C until glucose was completely consumed, and then heterologous expression was induced with galactose (3%, w/v) for 20 h (24).
Preparation of Cell Extracts, Cytosol, and Microsomes-- Cells harvested from the cultures by centrifugation were resuspended in Buffer A (100 mM potassium phosphate containing 20% glycerol, 1 mM reduced glutathione, and 0.5 mM EDTA) and homogenized with glass beads (0.45-0.5 mm in diameter) in a Braun disintegrator (Braun GmbH, Mesungen, Germany) operating at 1500 × g with 30-s bursts and carbon dioxide cooling. Cell extract was obtained as a supernatant by centrifuging cell homogenate at 1500 × g for 10 min. The extract was centrifuged at 10000 × g for 15 min to remove mitochondria as a pellet, and the resulting supernatant was centrifuged at 100,000 × g for 90 min to obtain microsomes as a pellet and cytosol as a supernatant. The microsomal pellet was resuspended in Buffer A using a Potter-Elvehjem homogenizer. Protein content in cell extract and microsomes was measured using the BCA protein estimation kit (Sigma) and bovine serum albumin as a standard (19, 24).
Purification of Soluble Yeast and Human CPR--
Soluble yeast
and human CPR purifications were carried out at 4 °C and a flow rate
of 1 ml/min as described previously, with few modifications, and
omitting detergent in the purification steps (25). The cytosol of JL20
expressing soluble CPR was precipitated with ammonium sulfate, and the
precipitate obtained with 40-65% saturated ammonium sulfate was
redissolved in 40 ml of Buffer B (10 mM potassium phosphate
buffer, pH 7.0, containing 1 µM FAD, 1 µM
FMN, 1 mM EDTA, and 10% (w/v) glycerol) and dialyzed
overnight against 4 liters of 10 mM Buffer B. The dialyzed
solution was centrifuged at 100,000 × g for 60 min to
remove precipitated material, and the supernatant was loaded onto a
hydroxyapatite column (2.0 × 10 cm) equilibrated with 10 mM Buffer B. The column was washed with 10 mM
Buffer B, and soluble CPR was eluted with a linear concentration
gradient of 80 ml of 10-180 mM Buffer B. The soluble CPR-containing fractions were pooled and directly loaded onto a column
(1.0 × 8 cm) of 2',5'-adenosine diphosphate agarose and washed
with 100 mM Buffer B, and soluble CPR was eluted with 100 mM Buffer B containing 5 mM
adenosine-2'-monophosphate (2'-AMP). The eluate containing soluble CPR
was dialyzed overnight against 2 liters of 100 mM Buffer B,
concentrated by ultrafiltration, and stored at 80 °C. Native yeast
and human CPR were purified according to published procedures (26).
In Vitro Ergosterol Biosynthesis-- The reaction mixture (1 ml) containing 924 µl of cell-free extract, 50 µl of cofactor solution (1 µmol of NADP, 1 µmol of NAD, 1 µmol of NADPH, 3 µmol of glucose 6-phosphate, 5 µmol of ATP, and 3 µmol of reduced glutathione dissolved in distilled water and adjusted to pH 7.0 with 10 M KOH), 15 µl of divalent cation solution (10 µl of 0.5 M MgCl2 and 5 µl of 0.4 M MnCl2), and 10 µl of [2-14C]mevalonate (0.25 µCi of 53 mCi/mmol) was incubated at 37 °C and 150 rpm. After 2 h of incubation, the reaction was stopped by adding 1 ml of freshly prepared saponification reagent (15% (w/v) KOH in 90% (v/v) ethanol), and the mixture was saponified by heating at 90 °C for 1 h. The nonsaponified sterols were extracted from the mixture twice with 3 ml of petroleum ether (boiling point 40-60 °C). The extracts were pooled, dried under nitrogen gas, and redissolved in 100 µl of petroleum ether. The nonsaponifiable sterols were applied to silica gel thin layer chromatography plates (ART 573, Merck) and developed using toluene:diethyl ether at a ratio of 9:1 (v/v). Radioactive sterols were localized by autoradiogram and excised for scintillation counting (27).
Reconstitution of CYP Activity-- The standard reaction mixtures contained purified S. cerevisiae CYP61, C. albicans and human CYP51 (0.5 nmol), 1 unit of either native yeast and human CPR or the N-terminal truncated soluble CPR forms, 13 nmol of the respective substrate (ergosta-5,7-dienol for S. cerevisiae CYP61 and dihydrolanosterol or 24-methylene-24, 25-dihydrolanosterol for human and C. albicans CYP51, respectively) dispersed in 80 nmol dilauroylphosphatidylcholine; the reaction volume was adjusted to 950 µl with 100 mM potassium phosphate buffer, pH 7.2. NADPH was added at a concentration of 1 mM to the mixture to start the reaction. C. albicans CYP51 was purified as described previously (28), and the human CYP51 was purified after GAL10 expression in yeast.3 All reactions were incubated at 37 °C for 20 min in a shaking water bath. Reactions were stopped by the addition of 3 ml of methanol, and the sterols were extracted and analyzed by gas chromatography/mass spectrometry as described above. Trimethylsilylated derivatives of sterol substrates and metabolites were clearly separated as two distinct peaks (for example, see Ref. 9). The conversion ratio was calculated from the areas of the two peaks, and the turnover (nmol of product formed/min/nmol of P450) was obtained from the amount of substrate added and the conversion ratio.
Spectrophotometric Measurements--
A Philips PU8800 UV/VIS
scanning spectrophotometer was used for all spectral studies.
Cytochrome c reductase activity was measured as described
previously (29). The absorption spectra of the purified yeast CPR
(33) was obtained by scanning from 300 to 700 nm (4). The ergosterol
content in the nonsaponifiable lipids was calculated on basis of its
molar extinction coefficient at 282 nm (Em = 11,900).
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RESULTS |
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Complementation Studies Using a cpr Gene
Disruptant--
The gene-disrupted strain
(CPR::URA3/JL20) was generated as described above
and had the phenotype described previously for a disruption using
LEU2 in the same way (13). Deletion of the CPR
gene led to a decrease in the levels of ergosterol. The ergosterol content in the cpr-disrupted strain was 4-fold lower than
that of the undisrupted strain (JL20). However, ergosterol was the major sterol in both the strains and accounted for about 90% of the
total sterols. In the cpr host strain, the ergosterol levels were restored to the levels in the CPR strain JL20 upon
yeast CPR and CPR (
33) expression (Table
I). Expression of these proteins in JL20
(CPR) did not alter the ergosterol content (data not
shown).
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Expression of Yeast and Human CPR Proteins in the Transformed
Strains--
For Western blot analysis of the expression and
localization of both yeast CPR and CPR (33), the cytosolic and
microsomal fractions of the cpr transformants expressing
these proteins were probed with anti-CPR (Fig.
1). The mobilities of the expressed yeast
proteins were consistent with their molecular masses (75 kDa for CPR
(
33) and 78 kDa for CPR). The Western blot data suggested that
GAL10-mediated expressed CPR (
33) and CPR proteins were localized in cytosolic and microsomal fractions, respectively. Heterologously expressed human native CPR and truncated human CPR
(
50) (the equivalent deletion to yeast CPR (
33)) and (
55) were
located in the microsomal and cytosolic fractions as determined spectrophotometrically. The yeast CPR contents were determined by using
the cytochrome c reduction assay and compared with the expressed human CPR, CPR (
50), and CPR (
55) forms. Yeast and human CPR contents in the cytosolic and microsomal fractions of the
transformants containing different expression vectors are shown in
Table II. The specific content and yield
of human CPR compared with yeast CPR was lower following heterologous
expression in S. cerevisiae. Furthermore, it was observed
that protein expression in the transformants expressing both soluble
yeast and human CPR were lower than those expressing full-length CPR.
No CPR was detected in the cytosolic fractions of the strain expressing
either yeast or human native CPR protein. However, in transformants
expressing soluble yeast and human CPR in the CPR host JL20,
the enzyme was observed in the cytosol as well as in the microsomes.
The amount of CPR present in the soluble CPR-expressing microsomes was
quite similar to that of microsomes from transformants containing the empty vector, YEp51, representing the basal endogenous CPR level in the
strain JL20. In addition, no CPR was detected in the microsomal fractions of the cpr strain transformed with either YEp51 or
CPR (
33):YEp51 after induction of expression, but it was observed in
the cytosolic fractions. The CPR level found in the cytosol was the
same as that observed in the cytosol of CPR strain
expressing CPR (
33):YEp51 (data not shown). These results
clearly indicated that the N-terminal truncated yeast and human CPR
localized in cytosol after expression similar to the previous reports
following trypsin cleavage experiments on mammalian CPR (3, 4).
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Purification of Soluble Truncated CPR--
Purification of yeast
CPR (33) to homogeneity by standard CPR methodology of ammonium
sulfate fractionation, hydroxyapatite, and 2',5'-adenosine diphosphate
agarose (affinity) chromatography yielded a 75-kDa protein. The
purified yeast CPR (
33) reduced cytochrome c at a rate of
24.3 µmol/min/mg of protein, compared with 36 µmol/min/mg of
protein for human CPR (
50) and CPR (
55). From this activity, the
CPR activity was estimated as 8 and 12 nmol/mg of protein for yeast and
human truncated CPR by assuming 1 nmol CPR reduces 3 µmol of
cytochrome c/min (26). The oxidized and reduced spectra of
purified yeast CPR (
33) protein were recorded to confirm the
presence of flavin cofactors (Fig. 2).
The oxidized spectrum of yeast CPR (
33) had peaks at 380 and 455 nm.
The spectrum obtained with the NADPH reduced CPR (
33) showed a
decrease in absorbance at 455 nm, an increase in absorbance at 380 nm,
and a broad absorption band between 550 and 650 nm characteristic of
the air-stable semiquinone form. These spectra were identical to those
reported for native microsomal yeast CPR (4).
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Catalytic Activities of Yeast CPR in Sterol
Biosynthesis--
In vitro ergosterol biosynthesis was
carried out by using the cell extracts of the various transformants of
the cpr host (Table III), an
assay applied for assessment of ergosterol biosynthesis inhibitors. The
amount of ergosterol synthesized in vitro was reduced by
about 3-fold upon CPR disruption when compared with the
parental strain. However, these changes were reverted when yeast CPR
and CPR (33) proteins were expressed in the cpr strain or
by adding purified yeast CPR (
33) to the cell extracts of cpr strain. The presence of the YEp51 expression plasmids in
the cpr strain did not alter the in vitro
ergosterol biosynthesis. To investigate further, reconstituted activity
was examined using yeast CPR (
33) protein together with different
purified CYP isozymes and comparison to the soluble human CPR forms.
Reconstitution was observed for the soluble yeast CPR (
33) when
added to samples containing S. cerevisiae CYP61, C. albicans, and human CYP51 (Table IV)
Reconstituted CYP activities for the soluble yeast CPR (
33) were not
significantly different from identical reactions using native yeast
CPR. In contrast, only native human CPR reconstituted CYP61, C. albicans CYP51, and human CYP51 activity. Soluble human CPR
(
50) (corresponding to yeast (
33)) and soluble human CPR(
55) (lacking the N terminus removed on tryptic digestion) both failed to
produce detectable activities.
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DISCUSSION |
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One of the earliest roles of CPR, as for the CYP superfamily, may
have been in sterol biosynthesis. CPR also functions in the squalene
epoxidation reaction (10), which precedes sterol 14-demethylation
undertaken by CYP51 (6), as well as sterol 22-desaturation undertaken
by CYP61, one of the last steps in the ergosterol biosynthesis pathway
(30).
The previous finding of hypersensitivity to ketoconazole on disruption of the gene encoding CPR was in many ways consistent with the concept of inefficient sterol biosynthesis, so that only a low dose of CYP51 inhibitor would arrest growth. We confirmed the observation of Sutter and Loper (13) of a 200-fold increased sensitivity in such a strain to ketoconazole,2 but we observed only a relatively small reduction in ergosterol synthesized to about 25% of the parent strain when compared by dry weight of cells. Analysis of sterols by gas chromatography and thin layer chromatography did not reveal accumulation of intermediates, such as lanosterol or ergosta-5,7-dienol, that are indicative of a block to enzyme activity at the steps in which CPR participates. This indicated that the electron donor system remaining in the cells is efficient, delivering both electrons for CYP activity.
Previously, the N terminus of mammalian CPR has been indicated to be the membrane anchor for the protein because trypsin cleavage was observed to release a soluble domain (5). However, this was not catalytically active in reconstituted systems, suggesting a role for the N terminus in interactions with CYP. Our studies indicate that the N-terminal 33 amino acids are important for membrane anchoring and targeting for yeast CPR, which contains a similar hydrophobic sequence. In contrast to previous studies on mammalian enzyme, this cytosolic protein is catalytically active and complements cpr-disrupted sterol biosynthesis on both expression and addition of purified enzyme to the in vitro system. This observation was confirmed in reconstitution studies with CYP61, in which normal activity was observed, as well as studies with C. albicans and human CYP51. This difference from mammalian studies was confirmed for the human enzyme and reflects a fundamental difference between the CPRs from different kingdoms. We can conclude that the yeast CPR membrane anchor is not required for supporting P450 activity in vivo or in vitro. Recently, the structure of rat CPR was resolved at 2.6 Å (31), and similar resolution of the yeast enzyme structure may help to elucidate the fundamental differences between the enzymes. For crystallization, the rat protein was produced in its soluble form after tryptic digestion, and the manner in which the hydrophobic domain allows interaction with CYP enzymes remains unclear. Presumably, the hydrophobic domain is important for the correct spatial interaction to allow electron transfer to CYP enzymes. Such interactions appear unimportant for yeast CPR in its interaction with fungal and mammalian CYP forms, and we are currently undertaking crystallization trials on soluble yeast CPR to resolve this and other issues.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Commonwealth Scholarship.
To whom correspondence should be addressed (present address):
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom. Tel.: 44-1970-622316; Fax: 44-1970-622350.
1 The abbreviations used are: CPR, cytochrome P450 oxidoreductase; CYP, cytochrome P450; PCR, polymerase chain reaction.
2 K. Venkateswarlu, D. C. Lamb, D. E. Kelly, N. J. Manning, and S. L. Kelly, unpublished observation.
3 D. C. Lamb, D. E. Kelly, M. Akhtar, M. R. Waterman, M. Stromstadt, D. Rozman, and S. L. Kelly, submitted for publication.
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REFERENCES |
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