Pollen tube attraction by the synergid cell.

In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

Cyclic nigerosyl-1,6-nigerose-based nanosponges: An innovative pH and time-controlled nanocarrier for improving cancer treatment.

The design and structural optimisation of a novel polysaccharide-based nanomaterial for the controlled and sustained release of doxorubicin are here reported. A cross-linked polymer was obtained by reacting a tetraglucose, named cyclic nigerosyl-1-6-nigerose (CNN), with pyromellitic dianhydride. The cross-linking reaction formed solid nanoparticles, named nanosponges, able to swell as a function of the pH. Nanoparticle sizes were reduced using High Pressure Homogenization, to obtain uniform nanosuspensions. Doxorubicin was incorporated into the CNN-nanosponges in a good extent. DSC and solid state NMR analyses proved the drug interaction with the polymer matrix. In vitro studies demonstrated pH-dependent slow and prolonged release kinetics of the drug from the nanoformulation. Doxorubicin-loaded CNN-nanosponges were easily internalized in A2780 cell line. They might considered an intracellular doxorubicin reservoir, able to slowly release the drug over time. CNN-nanosponges may be promising biocompatible nanocarriers for the sustained delivery of doxorubicin with potential localised application in cancer treatments.

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

Accurate and objective preoperative diagnosis of thyroid papillary carcinomas by reverse transcription-PCR detection of oncofetal fibronectin messenger RNA in fine-needle aspiration biopsies.

Recently, the restricted expression of oncofetal fibronectin mRNA was reported in thyroid papillary and anaplastic carcinomas. In this study, by extracting RNA from the leftover cells inside the needles used for fine-needle aspiration biopsy, we establish a new method for gene diagnosis of these carcinomas without further invasiveness to the patient (aspiration biopsy-reverse transcription-PCR, ABRP). RNA was extracted from 177 fine-needle aspiration biopsies of thyroid nodules that were suspicious for malignancy, and then the gene diagnoses made by reverse transciption-PCR detection of oncofetal fibronectin mRNA were compared with cytological diagnoses. Thirty-five (94.6%) of 37 samples that were diagnosed as papillary or anaplastic carcinomas by cytological examination showed a positive result by gene diagnosis, whereas only 4 (3.7%) of 109 samples that were cytologically diagnosed negative for both carcinomas showed a positive result. Among all of the cases, 50 patients underwent surgery, and a histological diagnosis was consequently made. The sensitivity and specificity of this method were 96.9 and 100%, respectively. A combined examination using both genetic and cytological approaches may contribute to a more precise preoperative diagnosis of papillary and anaplastic carcinomas.

A glial K(+) /Cl(-) cotransporter modifies temperature-evoked dynamics in Caenorhabditis elegans sensory neurons.

K(+) /Cl(-) cotransporters (KCCs) are known to be crucial in the control of neuronal electrochemical Cl(-) gradient. However, the role of these proteins in glial cells remains largely unexplored despite a number of studies showing expression of KCC proteins in glial cells of many species. Here, we show that the Caenorhabditis elegans K(+) /Cl(-) cotransporter KCC-3 is expressed in glial-like cells and regulates the thermosensory behavior through modifying temperature-evoked activity of a thermosensory neuron. Mutations in the kcc-3 gene were isolated from a genetic screen for mutants defective in thermotaxis. KCC-3 is expressed and functions in the amphid sheath glia that ensheathes the AFD neuron, a major thermosensory neuron known to be required for thermotaxis. A genetic analysis indicated that the regulation of the thermosensory behavior by KCC-3 is mediated through AFD, and we further show that KCC-3 in the amphid sheath glia regulates the dynamics of the AFD activity. Our results show a novel mechanism by which the glial KCC-3 protein non-cell autonomously modifies the stimulus-evoked activity of a sensory neuron and highlights the functional importance of glial KCC proteins in modulating the dynamics of a neural circuitry to control an animal behavior.

Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp-->Asn).

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63-->Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62-->Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.

Structure of an activity suppressing Fab fragment to cytochrome P450 aromatase: insights into the antibody-antigen interactions.

The crystal structure of a Fab fragment (Fab3-2C2) of a monoclonal antibody raised against aromatase cytochrome P450 P450arom) has been determined at 3.0 A resolution. P450arom is a membrane bound enzyme responsible for the catalysis of indrogens to estrogens, the process of aromatization, and hence has been implicated in hormone-dependent breast cancer. The Fab fragment of MAb3-2C2 IgG suppresses P450arom activity in a dose dependent manner. The Fab3-2C2 molecule crystallizes n the space group P2(1)2(1)2(1) with a unit cell of a= 154.89 A, b = 73.51 A, and c= 36.90 A. The crystal structure consists of a light and a heavy chain in the asymmetric unit, each characterized by the greek-key antiparallel beta barrel folding seen in all Fab structures. The average elbow angle between the two domains is 143 degrees. Modeling of the interactions between the variable domains of the antibody and a known model of P450arom maps the epitope to a region of the enzyme that is consistent with the available biochemical data and the activity-suppressing function of the antibody. The epitope mapping result is further supported by the inability of MAb3-2C2 IgG to suppress the activity of, or to interact with placental porcine P450arom, which is 81% identical (86% similar) to human P450arom but has a few key substitutions in the putative epitope region.

The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells.

In the isogamous green alga Chlamydomonas reinhardtii, the chloroplast genome is transmitted from the mt+ parent, while the mitochondrial genes are believed to be inherited from the mt- parent. Chloroplast nucleoids have been visualized by DAPI (4,6-diamidino-2-phenylindole) staining, and the preferential digestion of the mt- chloroplast nucleoids has been observed in young zygotes. However, the mitochondrial nucleoids have never been visualized, and their behavior is only deduced from genetic and biochemical studies. We discovered that the mitochondrial and chloroplast genomes can be visualized simultaneously in living cells, using the fluorescent dye SYBR Green I. The ability to visualize the mitochondrial and chloroplast genome in vivo permits the direct observation of the number, distribution and behavior of the chloroplast and mitochondrial nucleoids in young zygotes. Using this method, the biparental transmission of the mitochondrial genome was revealed.

Diverse functions of aromatase: O-deethylation of 7-ethoxycoumarin.

In studying the diverse functions of aromatase we found that purified and reconstituted aromatase also catalyzes O-deethylation of 7-ethoxycoumarin. Aromatase cytochrome P450 was purified from human term placentas by monoclonal antiaromatase P450 antibody-Sepharose 4B column chromatography. Kinetic analysis of the O-deethylation of 7-ethoxycoumarin by reconstituted aromatase showed Km of 200 microM, Vmax of 12.5 nmol.min-1.mg-1, and turnover rate of 1.06 min-1. 7-Ethoxycoumarin competitively inhibited androstenedione aromatization, the Ki was 180 microM. Fadrozole (CGS16949A), a specific competitive aromatase inhibitor, and MAb3-2C2, an antiaromatase P450 monoclonal antibody, inhibited both aromatase and 7-ethoxycoumarin O-deethylase activities dose responsively. The IC50 of Fadrozole was 33 nM for aromatase and 67 nM for 7-ethoxycoumarin O-deethylase. The IC50 of MAb3-2C2 was 1.1 micrograms IgG for aromatase and 4.0 micrograms IgG for 7-ethoxycoumarin O-deethylase. These results indicate that the two enzyme activities are catalyzed by the same active site of the cytochrome P450. Contrary to the previous postulate on the mechanism-based inactivation of microsomal aromatase by 4-androstene-3,6,17-trione, we found that with purified aromatase, both the initial 19-hydroxylase and the after lyase reactions are simultaneously inactivated by the steroid suicide inhibitor.

Preoperative diagnosis of medullary thyroid carcinoma by RT-PCR using RNA extracted from leftover cells within a needle used for fine needle aspiration biopsy.

Fine needle aspiration Biopsy (FNAB) is commonly used to diagnose thyroid tumors. In some clinical situations, however, accurate diagnosis requires a more objective method than cytological examination alone. Medullary thyroid carcinomas (MTC) derive from C cells in the thyroid and express some specific messenger RNAs (mRNA), such as those transcribed from the RET proto-oncogene, the calcitonin gene, and the gene for carcinoembryonic antigen (CEA), which usually do not exist in normal thyroid follicular cells or thyroid tumors of follicular epithelial descent. Recently, we established a new method for the molecular diagnosis of thyroid tumors without additional invasion to the patient by extracting RNA for RT-PCR from the leftover cells inside the needles used for fine needle aspiration biopsy (Aspiration Biopsy-Reverse Transcription-Polymerase Chain Reaction, ABRP). By applying the ABRP method to the detection of RET, calcitonin, and CEA mRNAs, an accurate molecular-based diagnosis for MTC maybe established as an adjunct to cytological diagnosis. In this study, 35 aspirates were obtained at the time of surgery from thyroid tumors, including 11 MTCs. The expression of these mRNAs in the leftover cells inside the needles used for the aspiration was then examined. Transcripts from all three genes were detected in the samples from all 11 MTCs, but none of these mRNAs were detected in the other tumors or normal thyroid tissues. Furthermore, MTC was preoperatively diagnosed in three patients by ABRP detection of these mRNAs, and these diagnoses were confirmed by subsequent cytological and histopathological analyses. Thus RT-PCR detection of RET, calcitonin, and CEA mRNAs in FNABs may be an efficient molecular adjunct for diagnosing MTC.

DNA staining for fluorescence and laser confocal microscopy.

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins.

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sjögren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M(r) of 55 kDa, specific heme content of 12.9 +/- 2.6 nmol.mg-1 (+/- SD, n = 4), reconstituted aromatase activity of 111 +/- 19 nmol.min-1.mg-1 and estradiol 2-hydroxylase activity of 5.85 +/- 1.23 nmol.min-1.mg-1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH beta-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 microM and Vmax of 8.9 nmol.min-1.mg-1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 microM, respectively, for testosterone aromatization, and 5.0 and 8.1 microM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 microM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1 beta-, and 2 beta-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid substrates to face their beta-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.

Diverse function of aromatase and the N-terminal sequence deleted form.

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Purification, characterization and crystallization of human placental estrone/dehydroepiandrosterone sulfatase, a membrane-bound enzyme of the endoplasmic reticulum.

Estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (ES/DHEAS) catalyzes the hydrolysis of E1 and DHEA-sulfates releasing unconjugated steroids. ES is a component of the three-enzyme system that has been implicated in intracrine biosynthesis of estradiol, hence, proliferation of hormone dependent breast tumors. ES is bound to the membrane of the endoplasmic reticulum, presumably through multiple transmembrane and other membrane anchoring segments. The highly hydrophobic nature of the enzyme has so far prevented its purification to homogeneity in quantities sufficient for crystallization. We report here the purification, biochemical characterization and crystallization of the full-length, active form of the enzyme from the membrane bound fraction of human placenta. Our results demonstrate that the key to successful purification and growth of diffraction quality crystals of this difficult membrane bound enzyme is the exploitation of optimal solubilization and detergent conditions to protect the structural and functional integrity of the molecule, thereby preventing nonspecific aggregation and other instabilities. This work paves the way for the first structural study of a membrane bound human sulfatase and subsequent rational design of inhibitors for use as anti-tumor agents.

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta.

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (> or = 20 cigarettes a day) than in normal and moderate smokers (< 20 cigarettes a day). At term, the mean aromatase activity and P-450arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.

Measurement of the expression of oncofetal fibronectin mRNA in thyroid carcinomas by competitive reverse transcription-polymerase chain reaction.

Abundant expression of oncofetal fibronectin mRNA has been observed in thyroid papillary and anaplastic carcinomas. In this study, we measured relative expression levels of oncofetal fibronectin mRNA in thyroid cancer tissues by competitive reverse transcription polymerase chain reaction (RT-PCR) using thyroglobulin mRNA as an internal control. By this method, all papillary and anaplastic carcinomas and 3 of 6 follicular carcinomas were distinguished from benign tissues, such as normal thyroid tissues, follicular adenomas, and adenomatous goiters. Furthermore, 2 anaplastic carcinomas were clearly distinguished from differentiated carcinomas. These results suggest the possibility of establishing a more accurate preoperative or postoperative diagnosis of papillary and anaplastic carcinomas by measuring the relative expression level of oncofetal fibronectin to thyroglobulin in thyroid tumors.

Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450.

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.

Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.