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Proc Natl Acad Sci U S A. Sep 27, 2005; 102(39): 14075–14079.
Published online Sep 19, 2005. doi:  10.1073/pnas.0505340102
PMCID: PMC1216831
From the Cover

Rapid inactivation of a moth pheromone


We have isolated, cloned, and expressed a male antennae-specific pheromone-degrading enzyme (PDE) [Antheraea polyphemus PDE (ApolPDE), formerly known as Sensillar Esterase] from the wild silkmoth, A. polyphemus, which seems essential for the rapid inactivation of pheromone during flight. The onset of enzymatic activity was detected at day 13 of the pupal stage with a peak at day 2 adult stage. De novo sequencing of ApolPDE, isolated from day 2 male antennae by multiple chromatographic steps, led to cDNA cloning. Purified recombinant ApolPDE, expressed by baculovirus, migrated with the same mobility as the native protein on both native polyacrylamide and isoelectric focusing gel electrophoresis. Concentration of ApolPDE (0.5 μM) in the sensillar lymph is ≈20,000 lower than that of a pheromone-binding protein. Native and recombinant ApolPDE showed comparable kinetic parameters, with turnover number similar to that of carboxypeptidase and substrate specificity slightly lower than that of acetylcholinesterase. The rapid inactivation of pheromone, even faster than previously estimated, is kinetically compatible with the temporal resolution required for sustained odorant-mediated flight in moths.

Keywords: Antheraea polyphemus, esterase, olfaction, pheromone-degrading enzyme, signal inactivation

To advertise their readiness to mate, female moths produce and release minute amounts of sex pheromones. With an exquisite olfactory system, male moths can detect these chemical signals remotely and take long-distance, odorant-mediated flights toward calling females. It is now known that pheromone-binding proteins (PBPs) (1) and odorant receptors (ORs) (24) are essential for the uptake, delivery, and detection of pheromones (5, 6). PBPs and ORs work with remarkable selectivity and sensitivity (7), but the molecular basis of signal termination is still terra incognita. While navigating through a pheromone plume, male moths encounter small pockets of pheromones separated by clean air spaces (8). To follow this pheromone trail, males must reset their olfactory system within milliseconds. Pheromone degradation in vitro by an antennal esterase (9), enzymatic inhibition in vivo (10, 11), and behavioral latency in pheromone response (12) suggests that this temporal resolution can be achieved by pheromone-degrading enzymes (PDEs). Because of low-level expression, limited sample size (≈60 nl per antenna), and a plethora of esterases in an insect's genome (13), PDE genes remained elusive for over two decades. Here, we report the isolation, cDNA cloning, expression, and kinetics of pheromone degradation by native and recombinant Antheraea polyphemus PDE (ApolPDE), an esterase expressed in pheromone-detecting sensilla in male antennae (1, 14), which can rapidly degrade physiologically relevant concentrations of pheromone. The ApolPDE identified here was previously characterized by Vogt et al. (9), then referred to as Sensilla Esterase.

Materials and Methods

Insects and Tissue Collection. Cocoons of A. polyphemus, purchased from Bill Oehlke (Pottersville, NJ) and Rinn Bozik (Studnicen Nachoda, Czech Republic), were kept at 4°C for at least 3 months to terminate diapause. In this publication, we defined day 0 pupal stage as the day when stored pupae were transferred to room temperature. Tissues were collected at each stage of the development from pupa to adult and extracted with ice-cold 10 mM Tris·HCl, pH 8 (Tris buffer). After centrifugation (12,000 × g,10 min, 4°C), the supernatant was concentrated and analyzed by 10% native PAGE, with proteins being visualized with α-/β-naphthyl acetate (9) and/or Coomassie brilliant blue R.

Isolation of ApolPDE. Nine hundred antennae of day 2 adult male moths were extracted with Tris buffer, centrifuged (12,000 × g, 10 min), and loaded on a DEAE (Toyopearl 650S, Tosoh, Tokyo; 16 mm × 16 cm; 5-ml bed volume) column. Proteins were eluted with Tris buffer containing increasing concentrations of NaCl (50–400 mM) and analyzed by 10% native PAGE (Fig. 3A). The fraction containing ApolPDE (250 mM) was diluted with Tris buffer and loaded on a MonoQ column (10/10, GE Healthcare, Piscataway, NJ). By elution with NaCl (0–800 mM; 2–42 min; 2 ml/min), esterase-active fractions were recovered at 24–27 min (Fig. 3B). These fractions were pooled, concentrated, and loaded on a calibrated gel filtration column (Superdex 75, GE Healthcare; 20 mM Tris·HCl, pH 8/150 mM NaCl; 0.5 ml/min). Esterase-active fractions were recovered at 23–27 min (60–64 kDa) (Fig. 3C). These fractions were pooled, diluted with Tris buffer, and further separated on a MonoQ column with a more shallow NaCl gradient (0–800 mM from 2 to 52 min). ApolPDE, recovered in fractions at 30–32 min, was analyzed by 10% native PAGE (Fig. 3D) and 10% SDS/PAGE (≈66 kDa, data not shown). To remove a contaminant later identified as protein disulfide isomerase, the sample was subjected to isoelectric focusing (Novex pH 3–7 isoelectric focusing gel, Invitrogen) (Fig. 4 A and B). Internal sequences were obtained by in-gel digestion and tandem MS (MS/MS) (Proteomic Research Services, Ann Arbor, MI).

Fig. 3.
Purification of ApolPDE by ion exchange and gel filtration chromatography. (A and B) Anion exchange chromatography (DEAE and MonoQ, respectively). (C) Gel filtration (Superdex 75). (D) MonoQ (more shallow gradient). The horizontal bars at the bottom of ...
Fig. 4.
Separation of native ApolPDE by isoelectric focusing. (A) Crude antennal extract (lane 1) and a small aliquot (≈1 ng) of the purified native ApolPDE (lane 2) were separated by isoelectric focusing and visualized by enzymatic activity. (B) pI standards ...

cDNA Cloning and RT-PCR. First-strand cDNA was synthesized from RNAs extracted from day 1 male antennae. PCR was carried out with degenerate primers based on amino acid sequences obtained by MS/MS: SG(K/Q)ATYVYK, SFA(I/L)HWVK, WDN(I/L)G(I/L)(I/L)ENK, PPTWYR or PPTGEYR, (I/L)AE(I/L)HA(I/L)K and (I/L)(F/M)(I/L)(F/M)D(I/L)GR. The full-length cDNA sequence was confirmed by using 24 independent clones. To determine expression pattern and tissue specificity, RT-PCR was carried out with gene-specific primers and cDNA prepared from identical animals as described in Figs. Figs.11 and and22.

Fig. 1.
Esterase activity in tissues of the wild silkmoth as visualized by α-/β-naphthyl acetate staining. ApolPDE (arrow) appears specifically in male antennae. MA, male antenna; I, integument; F, fat body; M, muscle; MG, midgut; MT, Malpighian ...
Fig. 2.
Development of male antennae and expression of ApolPDE during pupal-adult stages of the wild silkmoth. (A) Male antennae from pupae at 0, 2, 4, 6, 8, 10, 12, 13, and 14 days after incubation at room temperature and at day 0 adult. (Bar, 2 mm.) (B) To ...

Expression of Recombinant ApolPDE. After insertion of recognition sites of BamHI and XhoI at both ends of ApolPDE cDNA by PCR, the amplified DNA was ligated into corresponding sites of pFastBac1 (Invitrogen). The recombinant vector was transformed into MAX Efficiency Chemically Competent DH10Bac (Invitrogen). After inoculation of the positive transformants into LB medium containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, and 10 μg/ml tetracycline, recombinant bacmid DNA was purified with the Plasmid Mini Kit (Qiagen, Valencia, CA). The insertion of ApolPDE cDNA into the viral genome was confirmed by PCR. Recombinant virus (AcMNPV-ApolPDE) was obtained by transformation of the bacmid DNA into Sf21 cells with Celfectin (Invitrogen). Proteins showing esterase activity that comigrated with native ApolPDE were found in the medium of AcMNPV-ApolPDE-infected Sf21 cells but not in mock-infected cells. To purify the recombinant ApolPDE, the medium of AcMNPV-ApolPDE-infected Sf21 cells was harvested by centrifugation (500 × g, 5 min, 4°C) to remove debris, desalted (Centriprep YM-30, Millipore), and purified by the same protocol for the isolation of native ApolPDE.

Kinetic Studies. The reaction mixture (50 μl) consisted of one antenna equivalent of native or recombinant protein in 50 mM Tris·HCl, pH 7, in a glass insert deactivated by Silicote CL7 treatment (Kimble, Toledo, OH) and 1 μl of an ethanol solution of various concentrations of (E,Z)-6,11-hexadecadienyl acetate [E6Z11–16Ac]. These assays were performed with freshly (<4 h) isolated/extracted enzyme, which was kept at 4°C before use. The reaction was incubated at 25°C, quenched by injection of 2 μl of ethyl acetate, and transferred to a 100-μl V-vial (Wheaton, Millville, NJ). Substrate and product were extracted immediately with 20 μl of hexane (containing an internal standard, eicosyl acetate, Fuji Flavor, Tokyo) by vortexing for 30 s. After centrifugation (2,500 × g, 5 min, 4°C), the hexane layer was analyzed by GC and GC-MS. The GC detector was calibrated with a wide range of concentrations of hexane solutions of E6Z11-16Ac (Program Resources, Inc., Frederick, MD) and the corresponding alcohol, E6Z11-16OH (Chemtech, Amsterdam). Final concentrations of E6Z11-16Ac for the reactions (≈0.5–23 μM) were determined analytically by extracting the reaction at time 0. Velocity was calculated by the molar concentration of E6Z11-16OH per time (typically 1–2 min to consume <10% of substrate). These experiments were replicated at least five times for each pheromone concentration. Kinetic parameters (Vmax, Km) were determined by Lineweaver–Burk plots (double reciprocal, 1/v versus 1/[S]). The concentration of ApolPDE in an antenna was estimated by comparing (10% native PAGE) antennal esterase activity with calibrated curves generated with recombinant ApolPDE (nih image, Ver. 1.63). Protein concentrations were determined by using recombinant Bombyx mori pheromone-binding protein (BmorPBP) (15) as a standard.


Developmental Studies and Protein Isolation. Tissue specificity and the optimum time for protein isolation were determined on the basis of developmental studies. Two esterases were observed by native PAGE, the so-called integumental esterase (IE) [A. polyphemus IE (ApolIE)] (1) and a sensillar esterase (1, 14, 16), which we renamed ApolPDE (Fig. 1). ApolIE was detected in both olfactory and nonolfactory tissues (Fig. 1) and shown to be sex-indifferent (1, 16) (Fig. 2B), whereas ApolPDE was shown to be antennae-specific by analyzing extracts from antenna, integument, fat body, muscle, midgut, Malpighian tubules, hindgut, central nervous system, testis, and hemolymph (Fig. 1). Previously, this enzyme was named sensillar esterase because of its expression in pheromone-detecting sensilla (1, 14). No ApolPDE activity was detected in nonolfactory tissues at any stages of development (data not shown). ApolIE showed two apparent peaks at days 0–4 of the pupal stage and from day 14 of the pupal stage to day 0 of the adult stage (Fig. 2B). On the other hand, ApolPDE was expressed only in male antennae, with the onset of enzymatic activity detected at day 13 of the pupal stage (Fig. 2 B) coinciding with onset of antennae coloration (Fig. 2 A) and peak at day 2 adult stage (data not shown). ApolPDE was isolated from the extracts of 900 male antennae (corresponding to ≈53 μl of sensillar lymph; 900 antennae × 59 nl per antenna) by multiple steps of ion-exchange and gel filtration chromatography, with enzymatic activity monitored by native PAGE and α-/β-naphthyl acetate staining (Fig. 3). The purest fractions were pooled and subjected to isoelectric focusing electrophoresis to separate two bands comigrating on native PAGE (Fig. 3D). Esterase activity (Fig. 4A) was observed in the fast-migrating (pI, ≈3.5) band, which generated ≈500 ng of ApolPDE (Fig. 4B) for amino acid sequencing (MS/MS).

cDNA Cloning and ApolPDE Gene Expression. Although protein was extracted from antennae of day 2 male moths, the cDNA template for cloning was prepared from day 1 male antennae. Degenerate primers were designed on the basis of internal amino acid sequences obtained by MS/MS of the isolated protein. We obtained an 800-bp PCR product encoding two other independent amino acid sequences (LFIMDIGR and LAEIHALK) identified also by MS/MS of the native ApolPDE. With gene-specific primers, we cloned the full-length sequence of ApolPDE cDNA (AY866480) encoding 555 amino acid residues, including a 17-amino-acid residue-long signal peptide (see Fig. 6, which is published as supporting information on the PNAS web site). RT-PCR analyses indicated that the ApolPDE transcript specifically accumulated in male antenna but not in other control tissues, whereas actin transcripts were expressed in all experimental tissues (Fig. 6A). ApolPDE expression started at day 13 of the pupal stage and continued until the adult stage (Fig. 5B). Another olfactory gene, ApolPBP1 (17), started expression at day 14 of the pupal stage (Fig. 5B).

Fig. 5.
Gene expression detected by RT-PCR. (A) Expression in male antennae and lack of expression of ApolPDE in female antennae and control tissues. I, integument; F, fat body; M, muscle; MG, midgut; MT, Malpighian tubules; HG, hindgut; T, testis; MA, male antenna; ...

Concentration of ApolPDE in Sensillar Lymph. Recombinant ApolPDE was generated with a baculovirus expression vector in lepidopteran Sf21 cells. The native and purified recombinant enzymes migrated with the same mobility by native PAGE (see Fig. 7, which is published as supporting information on the PNAS web site) and on isoelectric focusing gels (data not shown), indicating that the recombinant PDE is identical to the native enzyme. Using calibrated curves of enzymatic activity and protein concentrations obtained with the recombinant esterase, the amount of native ApolPDE in a single antenna was estimated to be 2 ng. Considering the volume of sensillar lymph [59 nl, i.e., 1 pl per sensillum × 59,000 hairs per antenna (9, 14, 18, 19)] and the molecular weight of the enzyme [observed by SDS/PAGE, ≈66 kDa (data not shown); calculated 59.6 kDa], the concentration of the sensillar esterase was determined to be ≈0.5 μM, in agreement with a previous estimation of 1 μM (9).

Kinetics of Pheromone Inactivation. We studied the kinetics of pheromone degradation with crude antennal extracts and recombinant ApolPDE. Both native and recombinant enzymes showed comparable kinetic parameters (Km, 1.2 and 1.1 μM, respectively) and maximal velocity (Vmax, 75 and 66 nM per s, respectively) (see Fig. 8, which is published as supporting information on the PNAS web site). The Michaelis constants obtained by monitoring degradation of radiolabeled pheromone (9) are in the range of those reported here. As expected for an enzyme involved in the inactivation of pheromones, ApolPDE is fast (kcat, 127 s-1) and showed high substrate specificity (kcat/Km, 1 × 108 M-1·s-1). The enzyme has a turnover number (kcat) that is comparable to that of carboxypeptidase (20) (kcat, 100 s-1), higher than that of an insect hormone-degrading enzyme (juvenile hormone esterase; kcat, 1.6 s-1, estimated from ref. 21), and smaller than that of a neurotransmitter-degrading enzyme [acetylcholinesterase (20); kcat, 1,000 s-1]. The specific constant for ApolPDE indicates a substrate specificity slightly lower than that of acetylcholinesterase (20) (1.6 × 108 M-1·s-1) but larger than that of juvenile hormone esterase for juvenile hormone (3 × 107 M-1·s-1, estimated from ref. 21). The kinetics of pheromone degradation by ApolPDE was influenced by pH. Although fast at the bulk pH of the sensillar lymph, ApolPDE was very slow at low pH (see Fig. 9, which is published as supporting information on the PNAS web site).


ApolPDE is expressed specifically in male antennae of the wild silkmoth, A. polyphemus, in a concentration (0.5 μM) ≈20,000-fold lower than that of another olfactory protein, ApolPBP (10 mM) (9, 14). Given that A. polyphemus is at least 100-fold richer in olfactory proteins than other moths and beetles (data not shown), isolation of PDE in general is a daunting task, which can now be alleviated with a known PDE sequence. Previous attempts to identify PDEs by bioinformatics approaches (based on general esterases) have been unrewarding. A putative odorant-degrading enzyme, ApolODE, identified by us (16), is unlikely to be a PDE, as indicated by its expression pattern (Fig. 5B). The onset and basal expression of ApolODE at the early pupal stages are not consistent with an olfactory function limited to adult males, as expected for PDEs. A pattern unrelated to olfaction was also observed for the ubiquitous IE (1), ApolIE (Fig. 5B).

The remarkable sensitivity of the moth olfactory system has intrigued scientists for decades. The physiological and behavioral responses of moths to small amounts of pheromones (1 pg to 1 μg) (22), albeit striking, are only an underestimation of the sensitivity of the insect olfactory system. When stimulated for 1 s with 1 μg of pheromone (in a standard electrophysiology setup), male B. mori moths, for example, detect as little as 0.11 pg of the chemical signal given that only 1/60,000th of the pheromone amount loaded on a stimulus device is released per second, and of that, 1/150th is adsorbed on the antennae 5 cm away from the stimulus outlet (7). Under these physiological conditions (1 μg of stimulus), the concentration of pheromone in the sensillar lymph (≈6.6 nM) is <1% of Km, so that pheromone degradation essentially follows first-order kinetics (23). It has been suggested that the half-life of stray pheromone molecules in the sensillar lymph is 15 ms (9), but this is an underestimation, given that the enzyme efficiency (kcat, 127 s-1) is higher than that obtained with native enzyme isolated directly from gels (9) (kcat, ≈0.033 s-1). An antennae-specific aldehyde oxidase, isolated from Manduca sexta (24), was demonstrated to be even faster than the partially purified sensillar esterase from A. polyphemus (9). The rapid inactivation of a pheromone by ApolPDE is consistent with the temporal resolution required for sustained odorant-mediated flight through a pheromone plume (8). The findings presented here largely agree with and reconfirm those presented earlier by Vogt et al. (9) regarding this same enzyme. It seems, however, that localized low-pH environments generated by negatively charged surfaces on the dendrites (25) are essential to prevent “premature inactivation.” It is known that negatively charged chemical surfaces on cell membranes give rise to an electric surface potential, which in turn decreases the surface pH (26). These localized low-pH dendritic surfaces have been hypothesized to be involved in the fast delivery of pheromones (27) by a pH-dependent conformational change of a PBP (15, 2729). This “undocking” of pheromone may be possible because the enzyme is sluggish at low pH (see Fig. 9). Our in vitro system demonstrates that stray pheromone molecules (like those dissociated from the odorant receptors) are rapidly degraded by ApolPDE. Thus, the moth olfactory system can be reset by PDEs while navigating through clean air spaces in a pheromone plume.

Supplementary Material

Supporting Figures:


We thank A. M. Chen, V. P. Chiang, and T. I. Morgan for assistance in the isolation of proteins; S. G. Kamita for advice on protein expression; and Z. Syed, S. G. Kamita, M. Wogulis, and E. Lyons for valuable discussions. This work was supported by grants from the National Science Foundation (0234769) and National Research Initiative of the U.S. Department of Agriculture [U.S. Department of Agriculture–Cooperative State, Research, Education, and Extension Service (2003-35302-13648)].


Author contributions: W.S.L. designed research; Y.I. and W.S.L. performed research; Y.I. and W.S.L. analyzed data; and Y.I. and W.S.L. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PBPs, pheromone-binding proteins; PDEs, pheromone-degrading enzymes; ApolIE, A. polyphemus integumental esterase; ApolPDE, A. polyphemus PDE enzyme; MS/MS, tandem MS.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY866480).


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