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Antimicrobial Agents and Chemotherapy, August 2005, p. 3387-3395, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3387-3395.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human Lactoferricin Is Partially Folded in Aqueous Solution and Is Better Stabilized in a Membrane Mimetic Solvent

Howard N. Hunter,¹ A. Ross Demcoe,¹ Håvard Jenssen,² Tore J. Gutteberg,² and Hans J. Vogel^1*

Department of Biological Sciences, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta, Canada T2N 1N4,¹ Department of Medical Microbiology, University Hospital of North Norway, N-9038 Tromsø, Norway²

Received 13 February 2005/ Returned for modification 18 March 2005/ Accepted 15 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactoferricins are highly basic bioactive peptides that are released in the stomach through proteolytic cleavage of various lactoferrin proteins. Here we have determined the solution structure of human lactoferricin (LfcinH) by conventional two-dimensional nuclear magnetic resonance methods in both aqueous solution and a membrane mimetic solvent. Unlike the 25-residue bovine lactoferricin (LfcinB), which adopts a somewhat distorted antiparallel ß sheet, the longer LfcinH peptide shows a helical content from Gln14 to Lys29 in the membrane mimetic solvent but a nonexistent ß-sheet character in either the N- or C-terminal regions of the peptide. The helical characteristic of the LfcinH peptide resembles the conformation that this region adopts in the crystal structure of the intact protein. The LfcinH structure determined in aqueous solution displays a nascent helix in the form of a coiled conformation in the region from Gln14 to Lys29. Numerous hydrophobic interactions create the basis for the better-defined overall structure observed in the membrane mimetic solvent. The 49-residue LfcinH peptide isolated for these studies was found to be slightly longer than previously reported peptide preparations and was found to have an intact peptide bond between residues Ala11 and Val12. The distinct solution structures of LfcinH and LfcinB represent a novel difference in the physical properties of these two peptides, which contributes to their unique physiological activities.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The bactericidal effects of lactoferrin and lactoferricin (Lfcin) have been the subject of intensive study for at least two decades. Originally, the antimicrobial properties of intact lactoferrins were believed to be related to the iron-scavenging ability of this iron-binding protein (6, 43). Subsequent studies have shown, however, that potent antimicrobial properties reside in the highly basic N-terminal regions of these proteins, which are not involved in iron binding (8, 16). As a result, further study was given to the isolation of the lactoferricin peptide produced in vivo by digestion of bovine lactoferrin (27, 40). More recently, additional work has focused upon the identification of key antimicrobial segments and enhancement of the activity of the various shorter synthetic human lactoferricin (LfcinH) peptide analogs by using amino acid substitutions and various activity assays (7, 10, 11, 28, 30, 37, 42, 47). These studies have established not only that LfcinH has antimicrobial, antiviral, and antifungal activities but also that it is capable of stimulating the immune system and neutralizing endotoxin.

Other research has focused on determining how lactoferricin functions with specific target systems. For example, lactoferricin can effectively interact with porins (34) and DNA (39), bind to lipopolysaccharide and teichoic acid (5), reduce the charge potential at the bacterial membrane as well as the pH gradient (1), and display antiviral properties (2-4, 23, 24). Lactoferricin B has also been found to translocate into the bacterial cytoplasm (19), where it may exert its actions (38).

Independent of its mode of action, the initial step in the function of the Lfcin peptide involves the interaction with the cellular membrane. In this respect, knowledge of the preferred conformation of the peptide in solution may play an important role in understanding the ability of the peptide to interact with bacterial membranes. The structure of the 25-residue bovine lactoferricin (LfcinB) has been known to adopt a ß-sheet conformation in aqueous solution (21). This is markedly different from the structure of this region in the crystal structure; and this large conformational change, which involves the conversion from an alpha-helical region into a ß sheet, has been analyzed in detail by molecular dynamic simulations (48). Since LfcinB is more active as an antimicrobial peptide and is easier to obtain than LfcinH (41), historically more effort has been directed to the identification of the critical residues necessary for its potent antimicrobial activity. Although the conformation of shorter peptides derived from LfcinB has been studied in membrane mimetic conditions (32, 35), little work has been directed to date toward identifying a preferred conformation for the substantially longer LfcinH peptide in solution or in a membrane-bound form.

In the study described in this report, we have identified a preferred conformation of LfcinH both in aqueous solution and in a membrane mimetic solvent. The LfcinH structure does not resemble the regular solution structure determined for LfcinB. A comparison of the two structures aims to provide a better understanding of the behavior of the LfcinH peptide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactoferricin preparation and sequence analysis. LfcinH was prepared by pepsin digestion of native human lactoferrin purified from human milk (8), in collaboration with Tine Meierier (Oslo, Norway) and the Center for Food Technology (Queensland, Australia). The peptide was purified to 99.7% by reversed-phase high-pressure liquid chromatography on a Shimadzu PrepStar 8000 system with a C₁₈ column (22 by 250 mm; Vydac 218TP1022). The mass of lyophilized LfcinH was determined on a QSTAR Pulsar ESI/QqTOF mass spectrometer (PE Sciex, Canada). Portions of the amino acid sequence from LfcinH were verified in collaboration with the Biotechnology Centre of Oslo (University of Oslo, Oslo, Norway). First, LfcinH was subjected to reduction and alkylation (18), before digestion with trypsin for periods of over 4 h each, in 0.2 M NH₄HCO₃, pH 8.5, containing 2 M urea. Then, the tryptic peptides were purified on a reversed-phase high-pressure liquid chromatography system (Altex model 312; Uvikon LCD 725) on a C₁₈ column and were detected at 222 nm. The fractions were then evaporated and dissolved in formic acid, and the amino acid sequence was determined by standard Edman degradation with an Applied Biosystems 477A automatic sequence analyzer with an online 120A phenylthiohydantoin amino acid analyzer (12).

NMR spectroscopy. Altogether, the LfcinH peptide was investigated by nuclear magnetic resonance (NMR) spectroscopy under four different conditions, which required the preparation of four separate samples. The first sample was prepared by dissolving 2 to 3 mg of LfcinH peptide in 0.5 ml of 4:4:1 methanol-d₃-CDCl₃-H₂O (membrane mimetic solvent). After data acquisition, the solvent was evaporated, the sample was redissolved in 90:10% H₂O-D₂O, and the pH was adjusted to 3.5. Dodecylphosphatidylcholine (DPC) and sodium dodecyl sulfate (SDS) were added to two other aqueous samples, respectively, and the pH was adjusted to 4.0. The final concentrations of peptide in each sample was 1.40 x 10^–3 M, while the concentrations of DPC in one sample and SDS in another sample were 1.40 x 10^–1 and 1.26 x 10^–1 M, respectively.

All NMR spectra were acquired at 25°C on Bruker Avance 500- and 700-MHz NMR spectrometers equipped with either a 5-mm Cryo-probe (500 MHz; Bruker Analytische Messtechnik GmbH) or a 5-mm TBI three-axis gradient probe (700 MHz). The two-dimensional (2-D) total correlation (TOCSY) spectrum and the nuclear Overhauser enhancement (NOESY) spectrum were collected for the peptide in membrane mimetic solvent by using mixing times of 80 ms for the 2-D TOCSY and 250 ms for the 2-D NOESY spectrum. The NOESY spectra for the peptide in aqueous solution and with micelles were acquired with mixing times of 200 and 100 ms, respectively. Spectra were acquired with 2,048 x 512 to 600 datum points in the F2 and F1 dimensions by using spectral widths of 6,265 Hz at 500 MHz and 9,200 Hz at 700 MHz. Water suppression for the 2-D spectra was performed by using the excitation sculpting technique (22).

The 2-D TOCSY and NOESY NMR spectra were processed with the NMRPipe software package and analyzed with the NMRView 5.0.4 software package (14) on workstations operating with the Redhat 8.0 version of the Linux operating system. The 2-D data were zero filled once in each dimension and Fourier transformed with a shifted sine-bell function.

NMR diffusion data for 2 to 3 mg of peptide dissolved in 0.5 ml D₂O adjusted to pH 3.0 (uncorrected) were acquired and processed as described previously (20).

Structural calculations. The assignments of the peptide chemical shifts were determined by standard methods (46). Upon completion of the proton assignments, NOE-based distance restraints were collected from NOESY spectra and were automatically allocated to short-, medium-, and long-distance interactions based upon the peak volume. Broad dihedral restraints were used to confine the bond angles (except for Gly) to the allowed Ramanchandran space. The peptide structures were determined by using the programs CNS 1.0 and ARIA 1.2 (9, 33). ARIA calculations were initiated by using the default parameters in all runs except the final ARIA run, where the number of structures generated in the seventh and eighth iterations was increased to 40 and 100, respectively. Also, in the eighth iteration, the 20 lowest energy structures were used for statistical analysis. Molecular structures were viewed by using MOLMOL 2K.2 (26) and were analyzed by using PROCHECK 3.4 (29, 31). Residues recognized by MOLMOL as having potential hydrogen bonds in at least 60% of the structures were noted, and hydrogen bonds were introduced in the final ARIA calculation.

Protein structure accession numbers. Atomic coordinates and structure factors for LfcinH in aqueous and membrane mimetic solvents have been deposited in the Protein Data Bank (accession numbers 1Z6W and 1Z6V, respectively), Research Collaboratory for Structural Informatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence analysis. The original characterization (8) of LfcinH revealed that the disulfide cross-linked peptide comprises 47 residues, with one cleavage site occurring in the peptide chain. Completion of the initial NMR assignments for LfcinH suggested the presence of two additional C-terminal residues and the presence of a continuous polypeptide chain (see below). We therefore reinvestigated the sequence of the peptide before initiating structural calculations. The calculated mass for the isolated peptide with the Ala11-Val12 peptide linkage intact and two additional C-terminal residues, Gln48 and Ala49, is 5,738.4 Da, which compares favorably to the actual mass of 5,737.8 Da. Additional analysis of the peptide fragments identified in the trypsin digest is indicated in Fig. 1 and establishes beyond doubt the carboxy-terminal portion of the peptide. Edman degradation of these fragments also confirms the existence of an intact peptide bond between residues Ala11 and Val12. This peptide sequence for LfcinH has been confirmed by making independent digests of recombinant human lactoferrin at the Department of Biological Sciences, University of Calgary (data not shown); reduction of the disulfides in these preparations gave rise to one intact 49-residue peptide with an expected mass increase of 4 Da. The identification of two additional C-terminal residues and an intact peptide bond between Ala11 and Val12, as shown in Fig. 1, are not consistent with the amino acid sequence for LfcinH reported earlier (8).

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FIG. 1. Primary structure of LfcinH. Edman degradation was performed with fragments of the peptide obtained by trypsin digestion. Segments in bold were detected. The six N-terminal amino acids (GRRRRS) had previously been verified by N-terminal Edman degradation of the intact protein.

Structural NMR spectroscopy. The 2-D NOESY spectra of the aqueous sample with DPC gave only a few broad correlations which were not suitable for structure determination. The 2-D NOESY spectra for the sample in SDS gave a slightly greater number of correlations in the fingerprint region, but the quality of these correlations was still not acceptable for structural analysis. In addition, no amide-amide correlations were present in the 2-D NOESY spectrum with either type of micelle. The lack of a well-defined structure with either type of micelle is most likely caused by the conformational exchange that occurs on the NMR timescale. This type of conformational interconversion would explain why many of the correlations in the 2-D NOESY spectra were blurry and broad or not present at all.

The sample of LfcinH in aqueous solution gave numerous contours in the fingerprint, amide, and aliphatic regions of the 2-D NOESY spectrum, indicating that a preferred conformation had been adopted. Assignment of the correlations to protons from the peptide sequence yielded two unexpected observations. First, the 2-D NOESY spectrum indicated several correlations from the amide NH proton of Val12, including a correlation between the alpha proton of Ala11 and the amide NH proton of Val12. The presence of an amide proton for Val12, as well as these sequential correlations, would be observed only if the peptide bond between Ala11 and Val12 is intact. Second, there were two extra sets of correlations in the amide region corresponding to Gln48 and Ala49. These two findings indicate that the peptide bond between Ala11 and Val12 is not cleaved and that LfcinH is a 49-residue peptide with two additional C-terminal residues. The NMR data are consistent with the sequencing and mass spectrometry data reported above.

After assignment of all of the correlations in the NOESY spectra, it was noted that insufficient correlations were present to allow assignment of the large set of distance constraints necessary to properly define the relative position of each and every residue in the peptide. These types of constraints are usually obtained between residues that are more than one amino acid apart in the amino acid sequence, and they define the overall folding of the peptide (46). In the amide region nearly all of the correlations were between adjacent residues, and almost no longer-range correlations were present. The total number of constraints attributed to nonadjacent residues is shown in Fig. 2A. This type of analysis indicated three regions of relatively well defined structure: Ser6 to Val12, Thr18 to Gln24, and Pro33 to Ile38. Many of the correlations in the Ser6 to Val12 region involved interresidue contacts, primarily hydrophobic. Conversely, many of the interactions in the Thr18 to Gln24 portion were with the Pro33 to Ile38 portion and, again, were primarily hydrophobic in nature. Also, numerous interactions with a helical conformation were identified in the Thr18 to Gln24 segment. These interactions (d) between adjacent residues (i,i + 1) were mainly d_NN(i,i + 1) and d_N(i,i + 1) with weaker d_N(i,i + 2) and dß(i,i + 3) assignments.

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FIG. 2. (A) Graphical representation of the number of interresidue but nonadjacent NOEs for each amino acid in the LfcinH sequence in aqueous solution; (B) chemical shift index for LfcinH in aqueous solution.

Upon completion of the proton chemical shift assignments, initial insight into regular secondary structure can be gained by comparison of the alpha-proton chemical shifts to the chemical shift index (CSI) (45). For the LfcinH peptide there were insufficient numbers of sequential clusters of similar type to permit the assignment of regular structure, as shown in Fig. 2B. However, there were indications of a coiled region between Thr18 and Lys29. This same region is well known to form an helix in the intact protein.

Since the presence of DPC or SDS micelles with LfcinH did not provide conditions suitable for the formation of a regular structure which could be determined by NMR methods, a previously reported organic solvent system (4:4:1 methanol-chloroform-water) which mimics membrane systems was used (17, 36). The 2-D NOESY spectrum of the sample dissolved in the membrane mimetic solvent displayed many well-defined correlations. After complete assignment of the spectrum, it was evident that a helix was present between residues Gln14 and Met27. The interactions leading to the helical structure are seen in the sequential and medium-range ¹H-¹H NOEs shown in Fig. 3A. Except for regions of chemical shift degeneracy, all of the expected d_NN interactions were present. Included in these correlations were the amide proton of Val12 and its NOE interaction with amide proton Ala11, again confirming the presence of an intact peptide bond between the two residues. Shown in Fig. 3B is the CSI diagram for the alpha protons of each residue, which also indicates the helical structure between Gln14 and Lys29.

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FIG. 3. (A) 1H NMR summary indicating the interactions observed in the 2-D NOESY spectra for LfcinH in the membrane mimetic solvent. The thickness of the lines (strong, medium, and weak) indicates the intensity of the NOE interactions observed. Dashed lines indicate multiply assigned NOEs. Data are summarized from the final data report from the ARIA software. (B) The chemical shift index for LfcinH in chloroform-methanol-water. (C) Overlay of the 20 structures with the lowest energy conformation determined for LfcinH in membrane mimetic solvent.

There were also several interresidue interactions in the vicinity of the two disulfide bonds to confirm that the two expected cysteine linkages were present, consistent with the findings of mass spectrometry analysis. Furthermore, there were over 30 cross-strand interactions to confirm that the segments Cys20 to Met27 and Lys29 to Cys37 were arranged in a close to an antiparallel orientation. Although the peptide in the intact protein crystal structure displays a well-defined ß sheet between residues Val7 to Ala11 and Val35 to Lys39, no evidence of this type of regular structure was visible in the assigned NMR spectrum. Further evidence of the lack of a ß-sheet structure was seen by inspection of the amide proton correlations in the fingerprint region, which displayed ³J_NH- couplings that were too small to measure, making them less than the value of 9 Hz which is normal for ß strands. Finally, no contours were observed, which could be attributed to cross-strand interactions between the segments Val7 to Ala11 and Val35 to Lys39.

Structural calculations. The structural calculations of LfcinH in aqueous solution indicated a well-defined structure for regions of the peptide, as shown in Table 1. As expected, these regions primarily involved Ser6 to Val12, Thr18 to Gln24, and Pro33 to Ile38. The ribbon representation of the average energy-minimized structure is shown in Fig. 4A. This structure shows a helical region for Pro15 to Thr18, and the coiled backbone continues to Gln24. A turn at Lys29 and Val30 leads Val30 to Cys37 back in an antiparallel alignment to Cys20 to Gln24. However, except for the disulfide bridge from Cys10 to Cys47, there is no close association between Gly1 to Lys19 and Ile38 to Ala49.

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TABLE 1. Structural statistics for the final 20 structures from NMR data in aqueous and membrane mimetic solvents

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FIG. 4. Representations of calculated structures of LfcinH in aqueous solvent (A and B) and membrane mimetic solvent (C and D). (A and C) Ribbon diagram representations; (B and D) charge distributions on the surface of the peptides. Positive, negative, and neutral potentials are colored blue, red and white, respectively. This figure was produced by the MOLMOL program (26).

The NMR data from the sample in the membrane mimetic solvent mixture provided a significant increase in regular structure. The statistics from the 20 lowest energy conformations produced by ARIA are shown in Table 1, and the overlay of these structures is shown in Fig. 3C. The root mean square deviation (RMSD) fit of the data readily shows that a consistent helical structure is found between residues Gln14 and Lys29.

Figure 5 provides a comparison of the structure in the membrane mimetic solvent with the structure that the peptide assumes in the crystal structure of the intact protein (accession no. 1B0L in the Protein Data Bank). This comparison demonstrates that although the portion of the peptide from Gln14 to Lys29 has a propensity to form a helical character under membrane mimetic conditions, both the N- and C-terminal ends of the peptide do not assume the regular ß-sheet structure seen in the crystal structure and subsequently remain coiled when they are solvated.

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FIG. 5. Similar perspectives of ribbon representations of the calculated structures of LfcinH in aqueous solution (A), as isolated from the crystal structure of human lactoferrin (Protein Data Bank accession no. 1B0L) (B) and in the chloroform-methanol-water membrane mimetic solvent (C). The cysteine residues, tryptophan side chains, and disulfide bonds are shown in stick representation, while the Trp side chains are also highlighted. This figure was produced by the MOLMOL program (26).

Figure 4C and D shows a ribbon diagram along with the same view of the electrostatic surface plot of the LfcinH peptide in the membrane mimetic solvent. Comparison of Fig. 4C and D shows that the helical structure can accommodate the interaction of the charged side with the aqueous solvent. The surface plot indicates that the membrane mimetic environment of the organic solvent supports the amphipathic characteristics of antimicrobial peptides.

NMR diffusion. Several correlations were observed in the 2-D NOESY spectrum of the peptide in aqueous solution which were not consistent with the presence of a single structure. We therefore investigated whether LfcinH could form a dimer in this solution. Analysis of the diffusion experiments of the LfcinH peptide in D₂O indicated that the hydrodynamic radius of 18.4 Å was consistent with a nonspherical dimer, as the calculated spherical dimer radius is 17.9 Å (44). The presence of a dimer would account for additional correlations not consistent with a single structure. It is possible that the overlap of the hydrophobic cleft from two LfcinH molecules seen in Fig. 4D could give rise to additional intermolecular contacts which would account for the additional interactions seen in the 2-D NOESY spectrum.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The amino acid sequence of the LfcinH peptide isolated in this study shows that the peptide linkage between Ala11 and Val12 is intact and that the peptide obtained by pepsin digestion is 49 amino acids long, whereas the LfcinH sequence reported previously (8) is 47 residues. Since in our studies several different digests of the protein consistently resulted in the peptide shown in Fig. 1, it is possible that the previously reported peptide sequence (8) resulted from extended proteolysis or a difference in the source of the pepsin used for digestion. Taken together, the outcome of these studies indicates a need to characterize individual preparations of LFcinH by mass spectrometry. LfcinH does not adopt a preferred conformation with either SDS or DPC micelles that can be observed by ¹H NMR methods. However, the LfcinH peptide can be induced into a helical conformation when it is placed in a solvent known to simulate a lipid environment (17, 36). This difference in structure may be attributed to the fact that lactoferricin undergoes complete solvation by membrane mimetic solvent, whereas the lactoferricin molecule has an inability to adopt a single preferred conformation in the presence of SDS or DPC micelles. The membrane mimetic conditions indicate that the peptide has the ability to assume an amphipathic conformation suitable for the penetration and possible translocation of the peptide across a lipid membrane.

The unique structures determined in both the aqueous and the membrane mimetic solvents may suggest a more general method of regular structure formation for these amphipathic, antimicrobial peptides. For LfcinH, it would appear that the disulfide linkage between Cys20 and Cys37 provides the anchor for closure of the loop to bring the hydrophobic regions of Phe21 to Met27 into the proximity of Pro33 to Val35. Although the Cys20-Cys37 disulfide bond is not essential for antimicrobial activity (42), loop closure appears to be important for the structure observed in aqueous solution. These cross-strand hydrophobic interactions are also present both in the membrane mimetic environment and, apparently, in the crystal lattice of the intact human lactoferrin protein. In the aqueous environment, the favorable interaction of these hydrophobic regions would minimize contact with the polar solvent. By introduction of the peptide to a hydrophobic environment, like that provided by the membrane mimetic solvent, the coiled shape of Gln14 to Lys29 in aqueous solution can be further induced to a regular helical shape, resulting in an overall amphipathic structure. This change represents a progression from a nascent helix (15) in aqueous solution to a well-defined helix in membrane mimetic solvent.

We note with interest that in Fig. 2 most of the long-range interactions for LfcinH in aqueous solution are seen for the two Trp residues, Trp9 and Trp23. It is by now well established that the indole ring of Trp often gives rise to the formation of "native" and "nonnative" interactions in proteins that are in the unfolded state (13, 25). Thus, a similar clustering of hydrophobic contacts around Trp side chains, as seen here for LfcinH, has been observed in various denatured proteins. It highlights the important role that Trp side chains play along the folding pathways of proteins.

A comparison of the structures of human and bovine lactoferricin peptides in solution indicates a decisive difference in regular structure. Divergent from the ß-sheet structure of the 25-residue LfcinB previously determined in aqueous solution (21), LfcinH adopts at best only a coiled structure under similar conditions. Subsequently, the helical content of LfcinH increases in a membrane mimetic environment. Part of the reason for the lack of a well-defined structure in aqueous solution may be due to the much larger size of the LfcinH peptide compared with that of LfcinB or the lack of amino acid sequence homology, leading to differences in charge distribution and hydrophobic character. In the latter case, it could easily be visualized where the change of Ser17 in LfcinB to Pro34 in LfcinH would not favor the formation of the ß sheet across the peptide loop (21).

In conclusion, the difference in the three-dimensional structures of LfcinH and LfcinB introduces yet another interesting distinction between the physical properties of these two peptides, which may contribute to differences in their antimicrobial activities (41). Further studies are required to determine the precise residues responsible for the preferential formation of a ß-sheet structure in LfcinB and a helical structure in the same region of LfcinH.

 
This work was supported by an operating grant from the Canadian Institute of Health Research to H.J.V. H.J.V. holds a senior scientist fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). The NMR Cryo-probe was purchased through grants from AHFMR, the Alberta Science and Research Authority, the Alberta Intellectual Infrastructure Partnership Program, and the Canada Foundation for Innovation

Insightful discussions with Evan Haney and Gilles Lajoie are gratefully acknowledged.

 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Calgary, 2500 University N.W., Calgary, Alberta, Canada T2N 1N4. Phone: (403) 220-6006. Fax: (403) 289-9311. E-mail: vogel{at}ucalgary.ca.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, August 2005, p. 3387-3395, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3387-3395.2005
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