| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,1,2
* Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA;
Department of Hand Surgery, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China;
Confocal Microscopy Center, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA; and
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA
1Correspondence: E-mail: twang40{at}jhmi.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: laminin receptor • extracellular matrix • microglia • neuron • MPP+ • Parkinson’s disease
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
]. Recent studies have indicated that inflammation plays important roles in neurodegenerative diseases such as PD and Alzheimer’s disease (2
, 3)
. After the initial injury, factors released from damaged neurons induce reactive gliosis, especially microgliosis, which is the hallmark of inflammation in the brain (4)
. Reactive microgliosis may enhance the neurotoxicity by producing excessive proinflammatory factors, including cytotoxic superoxide, NO, and proinflammatory cytokines (5
6
7)
. This microglia-dependent inflammatory process may underlie the progressive and self-propelling nature of neurodegenerative diseases such as PD.
Though the mechanism for initiation of microgliosis is not fully understood, it is suggested that the ECM plays a role in neuroinflammation. Expression of matrix metalloproteinases (MMPs) was reported in PD and other inflammation-related neurodegenerative diseases (8
, 9)
. ECM molecules, such as laminin and fibronectin, are substrates of MMPs and can be modified or cleaved by MMPs to release soluble forms. The soluble ECM molecules may regulate microglial functions (10)
. ECM is also important for the adhesion and migration of microglia. ECM molecules bind to the cell membrane through receptors, including integrin receptors and nonintegrin receptors, to regulate cell activities. A high-affinity 67 kDa nonintegrin laminin receptor (LR) has recently been reported to regulate monocyte functions (11)
. LR is a receptor for several ECM molecules (laminin, fibronectin, elastin) as well as prion protein (12
13
14)
. LR plays important roles in mediating the interactions between ECM and cells, such as cell adhesion, migration, proliferation, and differentiation (11
, 15
, 16)
. The induction of LR on transformed cells membrane was claimed a marker of the cell metastatic potential (17)
. LR expression was also observed in inflammation, often along with other inflammation-related factors such as CD14, CD11b/CD18 (leukocyte receptor for C3bi), fibrinogen, and endotoxin (11
, 18)
. Expression of LR can also be increased by its ligands such as laminin (19)
. LR expression in inflammatory cells is believed to play pivotal roles in mediating the immune process and cell differentiation (11)
. A mechanism involving LR-regulated growth factor signaling has also been reported (20)
.
Therefore, we speculated that LR might be expressed in microglia during neuroinflammation; by regulating microglia adhesion and migration, LR may mediate microgliosis and subsequent DA neurotoxicity. To test this hypothesis, we used 1-methyl-4-phenylpyridinium (MPP+) -treated mouse primary mesencephalic neuron-glia cultures as an in vitro PD model and observed that LR expression in microglia is increased by MPP+, and inhibition of LR results in attenuated microgliosis and DA neurodegeneration.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals
Timed pregnant C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).
Cell cultures
Primary mice mesencephalic neuron-glia cultures were prepared following our published protocol (21)
. Briefly, after the ventral mesencephalic tissues were removed and dissociated by a mechanical triturating, cells were seeded at 3 x 105/well to 48-well culture plates or 1.5 x 105/well to 96-well culture plates precoated with poly-D-lysine (20 µg/ml) and maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air in 0.25 ml maintenance medium, which consisted of minimum essential medium (MEM), 10% FBS and 10% HS, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. Fresh medium was added to each well 3 days later. Seven-day-old cultures were used for treatment. Immunocytochemical (ICC) analysis indicated that at the time of treatment the cultures were made up of 
10% microglia, 50% astroglia, and 40% neurons, of which 1–2% were TH-immunoreactive (TH-IR) neurons.
Microglia-enriched cultures were prepared from the whole brains of 1 or 2-day-old mice, as described previously (22)
. Briefly, brain tissues, devoid of meninges and blood vessels, were dissociated by a mechanical trituration. The isolated cells (5x107) were seeded in 150 cm2 culture flasks in Dulbecco’s modified Eagle medium/F12 containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Fresh medium was changed 4 days later. When reaching confluence (12–14 days), microglia were separated from astroglia by shaking the flasks for 5 h. Purity of the microglia-enriched cultures was >98%, as determined by ICC staining.
Primary microglia-depleted cultures were prepared by adding 1 µM leucine methyl ester into the primary neuron-glia cultures 24 h after the initial seeding. Fresh medium was changed 2 days later. Seven-day-old cultures were used for treatment. At the time of treatment, ICC analysis indicated that the microglial composition was <0.1%.
Immunocytochemistry
Neurons were stained with the Ab against Neu-N. Microglia were stained with rat mAb raised against F4/80 antigen, and astroglia were stained with the Ab against GFAP, an intermediate filament protein whose synthesis is restricted to astroglia. DA neurons were detected with the polyclonal antibody (pAb) against TH. LR expression was stained with the mAb against LR.
Immunostaining was performed as described previously (23)
. Briefly, 3.7% paraformaldehyde fixed cells were washed twice with PBS, then treated with 1% hydrogen peroxide for 10 min. After washing with PBS, cells were incubated for 40 min with blocking solution (PBS containing 1% BSA, 0.4% Triton X-100, and 4% appropriate serum: normal horse serum for Neu-N, LR and F4/80 staining and normal goat serum for TH and GFAP staining). The cultures were incubated overnight at 4°C with the primary Ab diluted in DAKO Ab diluent. After rinsing three times for 10 min each in PBS, the cultures were incubated for 1 h with PBS containing 0.3% Triton X-100 and the appropriate biotinylated secondary Ab (horse antimouse Ab, 1:250, for Neu-N and LR; goat anti-rabbit Ab, 1:250, for TH or GFAP; mouse antirat Ab, 1:500, for F4/80). After washing three times with PBS, the cultures were incubated for 1 h with the Vectastain ABC reagents diluted in PBS containing 0.3% Triton X-100. After washing three times with PBS, the bound complex was visualized by incubating cultures with 3,3'-diaminobenzidine and urea-hydrogen peroxide tablets (Sigma) dissolved in water. Images were recorded with a CCD camera and the MetaMorph software (Universal Imaging Systems, West Chester, PA, USA).
[3H]DA uptake assay
Cultures were incubated for 20 min at 37°C with [3H]DA (1 µM) in Krebs-Ringer buffer (16 mM NaH2PO4, 16 mM Na2HPO4, 119 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.3 mM EDTA, pH 7.4). After washing with ice-cold Krebs-Ringer buffer three times, the cells were collected in NaOH (1 N). Radioactivity was determined with a liquid scintillation counter (24)
. Nonspecific DA uptake was determined in the presence of 10 µM mazindol.
LR distribution in primary neuron-glia cultures
The distribution of LR expression in primary neuron-glia cultures was determined using confocal microscopy. Briefly, primary neuron-glia cells (1x106/well) were cultured on cover slips. After treatment with MPP+ (0.5 µM) for 48 h, the cells were fixed with 4% formaldehyde containing 0.1% Triton X-100 in PBS for 20 min at room temperature. After washing three times with PBS, cells were permeabilized with 2.5% triton X-100 in PBS for 10 min. After washing, the cover slips were blocked with 0.5% gelatin and 5% goat serum in PBS for 30 min at room temperature. Then the cells were incubated with mAb against LR (1:100) and Alexa 647 conjugated rat antimouse F4/80 (1:100, Caltag Laboratories, Burlingame, CA, USA) in PBS containing 0.1% gelatin and 1% goat serum at room temperature for 1 h. After washing three times with PBS containing 0.1% gelatin and 1% goat serum, the cells were incubated with Alexa fluor 488 rabbit antimouse Ab (1:100, Molecular Probes, Eugene, OR, USA) in PBS containing 0.1% gelatin and 1% goat serum for 1 h. A laser scanning confocal microscope (LSM 510 mounted on Axiovert 100M microscope, Carl Zeiss, Inc.) was used to obtain the immunofluorescence and differential interference contrast (DIC) images. The images were obtained using the 633 nM laser line for Alexa fluor 647 and the 488 nm line for Alexa fluor 488 and a Zeiss C-Apo 40x N.A. = 1.2 water immersion objective.
Phagocytosis
Primary neuron-glia cultures in 96-well culture plates were treated with MPP+. The phagocytosis was evaluated 48 h later by using a FluoSpheres Fluorescent Microspheres kit (Molecular Probes) according to the direction of the manufacturer. Briefly, the fluorescent microspheres were added into the cultures for 4 h, then the fluorescence was read at 485 nm for excitation and 530 nm for emission on a SpectraMax GEMINI XS fluorescence microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Confocal microscopy was used to determine the type of cells that phagocytizes the fluorescent microspheres. Briefly, after 4 h of the incubation with fluorescent microspheres, primary cultures on the cover slips were washed with PBS and fixed with 4% formaldehyde containing 0.1% Triton X-100 in PBS for 20 min at room temperature. The cells were stained with Alexa 647 conjugated rat antimouse F4/80 as described above. The images were obtained using the 633 nm laser line for Alexa fluor 647 and the 488 nm line for Alexa fluor 488 and a Zeiss C-Apo 40x N.A. = 1.2 water immersion objective.
Superoxide production
Superoxide production in primary neuron-glia cultures after MPP+ (0.5 µM) treatment with and without LR-Ab was measured with the WST-1 assay (25)
. Primary neuron-glia cultures (1.5x105/well) were grown in 96-well plates. For the superoxide assay, cultures were washed twice with Hanks’ balanced salt solution (HBSS), then incubated in 200 µl of 1 mM WST-1 in HBSS. The absorbance at 450 nm was read for 30 min at 37°C with a SpectraMax Plus microplate spectrophotometer (Molecular Devices). The amount of superoxide production was determined as the increase of absorbance in 30 min and expressed as percentage of the control cultures.
Western blot
After treatment with MPP+ for 24 h, primary neuron-glia cells were collected and lysed for Western blot for LR. Protein concentration was determined with the bicinchoninic acid assay (BCA) (Pierce, Rockford, IL, USA) following the manufacturer’s guide. Equal amounts of protein (20 µg per lane) were separated by NuPAGETM gel (NOVEX, San Diego, CA, USA) and transferred to PVDF membranes (NOVEX). Membranes were blocked with 10% skim milk and incubated with polyclonal anti-LR Ab (ab711, 1:1000) for 1 h at 25°C. Peroxidase-linked anti-rabbit IgG (1:5000;1 h at 25°C) and enhanced chemiluminescence (ECL)+Plus reagents (Amersham, Piscataway, NJ, USA) were used as a detection system. The optical density of the bands was measured with a model GS-700 Imaging densitometer (Bio-Rad, Hercules, CA, USA).
Cell viability assay
The cytotoxic effects of various agents on the viability of microglia were evaluated with MTT assay (23)
. After treatment with MPP+ and corresponding dilutions of LR-Ab for 48 h, 5 mg/ml MTT was added to the microglia-enriched cultures. Cells were incubated for another 2 h. The supernatant was removed and 100 µl DMSO was added to each well to dissolve the formed formazan. Absorbance was read at 550 nm.
Statistical analysis
Data are expressed as mean ± SE. Statistical significance was determined using an ANOVA, followed by the Bonferroni’s t test using the JMP program (SAS Institute, Cary, NC, USA). A value of P < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, MPP+ treatment significantly increased LR expression in neuron-glia cultures. The LR-immunoreactive (LR-IR) positive cells showed irregular shapes that are usually seen in activated microglia (Fig. 1A
); Western blot assay showed that MPP+ increased the production of LR in a dose-dependent manner (Fig. 1B
). These observations indicate that MPP+ increased LR expression in neuron-glia cultures
|
MPP+ increased microglial LR expression in neuron-glia cultures
LR-IR positive cells showed irregular shapes indicating activated microglia, suggesting that an MPP+-induced increase in LR expression is mainly in activated microglia. To discern the identity of cells up-regulating LR, we used different cultures, including neuron-glia, microglia-enriched and microglia-depleted neuron-astroglia cultures. These cultures were treated with 0.5 µM MPP+ for 24 h, then LR ICC was performed. As shown in Fig. 2
, LR expression increased in neuron-glia cultures but not in microglia-enriched or microglia-depleted cultures (neurons and astroglia cultures) after MPP+ treatment (Fig. 2A
), indicating that neurons and microglia are both necessary for MPP+-induced up-regulation of LR. Furthermore, confocal microscopic examination showed that LR expression was localized in activated microglia, which were positively stained with F4/80 (Fig. 2B
). These findings indicate that MPP+-increased LR expression is through reactive microgliosis.
|
LR antibody and YIGSR attenuated MPP+-induced DA neurotoxicity
To determine whether LR activation mediated MPP+-induced DA neurotoxicity, LR-Ab (ab711, against the C-terminal domain) and pentapeptide YIGSR (competitively blocks LR-laminin binding) were applied with MPP+ in the neuron-glia cultures. A pAb (ab2508) that recognizes the intracellular region of the LR (SGALDVLQ) was used as a negative control. A DA uptake assay for DA neuronal functions and TH ICC assays for cell morphology and counts were performed 4 days later in control and YIGSR-treated cultures. As shown in Fig. 3
, MPP+ (0.5 µM) treatment significantly reduced DA uptake in both neuron-glia and neuron-enriched cultures. Treatment with LR-Ab (Fig. 3A
) or YIGSR (Fig. 3B
) significantly attenuated MPP+-induced reduction of DA uptake in mixed neuron-microglia but not in neuron-enriched cultures. LR-Ab or YIGSR alone did not show neuroprotective effect in control and Ab2508 failed to show any protective effect in cultures (data not shown). TH ICC showed that MPP+ treatment decreased TH-IR cell numbers and resulted in fragmented and shortened TH-IR dendrites; YIGSR also significantly attenuated MPP+-induced cell loss (Fig. 3C
) and morphological change (Fig. 3D
) of TH-IR neurons in mixed neuron-microglia cultures by ICC. These results suggest that activation of LR on microglia plays important roles in mediating MPP+-induced DA neurotoxicity and this effect may be related to cell-ECM adhesion, which is regulated through LR.
|
LR antibody and YIGSR treatment attenuated MPP+-induced microgliosis
As MPP+-induced LR expression is mainly on activated microglia, we speculate that LR expression may be related to microglial activation or reactive microgliosis. To determine whether LR plays a role in mediating MPP+-induced microgliosis, polyclonal LR-Ab (ab711) (1:250) and YIGSR were added with MPP+ in the neuron-glia cultures for 24 h. Microgliosis were then examined by F4/80 ICC. As shown in Fig. 4
, after 24 h MPP+ treatment significantly increased numbers of F4/80-IR cells in neuron-glia cultures, which were attenuated by cotreatment with LR-Ab or YIGSR. This observation indicates that LR activation plays important role in mediating MPP+-induced microgliosis.
|
LR-Ab treatment attenuated MPP+-induced microglial phagocytosis in neuron-glia cultures
Phagocytosis, an important function of activated microglia, was used in our study as another index for microgliosis. Neuron-glia cultures were seeded in a 96-well plate, then MPP+ with/without LR-Ab was added in the media for 48 h. Phagocytosis activity was evaluated by adding fluorescent microspheres. As shown in Fig. 5
, MPP+ (0.5 µM) treatment in neuron-glia cultures for 48 h significantly increased the fluorescence compared to control cultures, whereas cotreatment with LR-Ab711 significantly attenuated the MPP+-increased fluorescence in a dose-dependent manner (Fig 5A
). Further confocal microscopic studies indicated that the cells containing fluorescent microspheres are also F4/80 positive stained (Fig. 5B
). These observations indicate that LR-Ab also significantly attenuated MPP+-induced microglial phagocytosis.
|
LR-Ab treatment attenuated MPP+-induced superoxide production in neuron-glia cultures
Previous work from our laboratory demonstrated that superoxide production as a result of microgliosis plays an important role in mediating MPP+-induced DA neurodegeneration (5)
. To investigate whether LR-Ab attenuated microgliosis also results in decreased superoxide production, superoxide production in the MPP+-treated primary neuron-glia cultures was detected. As shown in Fig. 6
, MPP+ treatment for 4 days significantly increased superoxide production, which was significantly decreased by cotreatment with LR-Ab in a dose-dependent manner. Thus, LR-regulated microgliosis are at least partly responsible for the MPP+-induced superoxide production.
|
Exogenous laminin induced microglial activation and DA neurodegeneration in neuron-glia cultures
To determine whether LR-laminin binding is the key event resulting in LR-involved neurodegeneration, recombinant laminin (1–10 µg/ml) was added to the mixed neuron-glia cultures. F4/80 staining was performed 24 h after treatment to determine the microglial activation, and DA uptake assay was conducted for neurotoxicity 4 days later. As shown in Fig. 7
, laminin induced microglial activation at 24 h as characterized by the increased number of F4/80-IR microglia (Fig. 7A
). Laminin also induced DA neurotoxicity as shown by the decreased DA uptake (Fig. 7B
). LR-Ab treatment partially attenuated laminin-induced microglia activation and neurodegeneration (Fig. 7A, B
). These observations suggest laminin-LR interaction is a possible mechanism for LR-regulated microgliosis and subsequent DA neurodegeneration.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
After the initial onset, the chronic and long-term progressive processes of the neurodegenerative diseases, including PD, are unknown. The existence of persistent reactive microgliosis and inflammatory process provides a mechanism that may explain the pathological process of the progressive nature. However, the lack of understanding of the specific mechanisms initiating the microgliosis has dampened the effort for developing new therapeutic approaches targeting microgliosis. Nevertheless, it has been suggested that interactions with ECM are necessary for macrophage and microglia functional activity (26
, 27)
. The influence of ECM proteins, including laminin and fibronectin, on microglia differentiation and activation has been reported in cell cultures (10
, 28)
. It is also reported that proinflammatory factors, including TNF, significantly increase microglial adhesion to laminin through integrin 
6ß1, a well-characterized laminin receptor (29)
with a close relationship to LR. These findings suggest that microglial adhesion to laminin may play a fundamental role in determining the extent of microglial infiltration into and retention at the site of injury. A previous study using adult rat (30)
has indicated that LR expression in brain (mainly in neurons and astroglia) increases with the maturation of the brain. In contrast, our primary neuron-glia mixed cultures derived from mouse embryo show no detectable LR expression in either neurons or astroglia (Fig. 2)
. This characteristic makes these cultures highly suitable for us to address the role of microglia in LR expression and subsequent neurotoxicity. Our findings that LR expression is only increased in microgliosis and blocking it with Ab or YIGSR attenuates microgliosis suggest LR is critical in regulating microglial function in the pathogenesis of inflammation-mediated neurotoxicity.
To further our study of the LR-mediated microgliosis, two important indexes for microgliosis—superoxide production and phagocytosis—were evaluated. Superoxide plays dual roles in mediating inflammation-related neurodegeneration. First, superoxide anion or peroxynitrite, a product from reaction of superoxide and NO, is directly toxic to neurons (31)
. Second, superoxide production in microglia may further activate microglia by increasing the production of other proinflammatory factors such as TNF and prostaglandins (22
, 32
, 33)
. Our observation that LR-Ab attenuated superoxide production suggests a possible regulatory role of LR on the NADPH oxidase, the predominant superoxide-producing enzyme in phagocytes. Our previous work indicated that microglial NADPH oxidase is responsible for superoxide production, which is critical for microglial activation and reactive microgliosis (5)
. In addition, NADPH oxidase can be activated by soluble elastin and prion protein (34
, 35)
, both of which are binding ligands for LR. Another function of activated microgliosis is phagocytosis. Besides scavenging the debris of damaged neurons, recent studies have suggested that phagocytosis may actively participate in neurodegeneration (36)
. Our observation that LR-Ab attenuated MPP+-increased microglial phagocytosis is further evidence for the importance of LR on microgliosis. LR-mediated adhesion, which is necessary for morphological changes of functional microglia during phagocytosis, may underscore its regulatory role of phagocytosis.
Although we have not detected the soluble laminin increase after MPP+ treatment in our system due to the detection limitation of commercial available kits, it is reasonable that because of the increased activity of MMPs (8
, 9)
in this PD model, there is an increase of soluble ECM macromolecules in the microenvironment around microglia. Since soluble ECM fragments such as soluble laminin can increase LR expression (19)
, their presence may be the reason for the initial increase of the LR expression in microglia. Furthermore, as it has been shown that activated LR can increase laminin degradation and the release of soluble laminin fragments (37)
, the latter may result in further LR expression in microglia. This would result in a self-propelling microglial activation, which may underlie the prolonged chronic process in PD. The actual factors that lead to LR expression need further study.
It is notable that LR may function in conjunction with other integrin receptors such as integrin 6ß1 and 6ß4 (38
, 39)
. The observation that LR-Ab only partially attenuates laminin-induced neurotoxicity also indicates that factors besides LR also play a role in mediating laminin-induced neurotoxicity. Because of the limited size of the intracellular domain, if any, of LR, it may require interaction with other integrin factors to induce intracellular signaling. The interaction between LR and 6 is critical as some reports have referred to LR as an auxiliary molecule for 6 function and indicate the existence of a possible receptor complex consisting of both (39
, 40)
. Furthermore, these two receptors are usually colocalized and 6ß1 is necessary for the membrane exportation of LR (19
, 38)
. The relationship between LR and integrin receptors and its effect on microgliosis are worth further study.
In summary, our observations suggest that by regulating cell-extracellular matrix interactions, LR plays important roles in mediating microgliosis and subsequent DA neurotoxicity. Laminin is a potential ligand for activating this LR. As microglial activation has been linked with a broad range of neuropathological processes, our observation that microgliosis is regulated by LR activation indicates that LR-mediated microgliosis may also play an important role in other neurological disorders besides PD. Therefore, laminin/LR may be a potential target for developing new therapeutic drugs against neurodegenerative disorders such as PD.
|
FOOTNOTES |
|---|
Received for publication September 12, 2005. Accepted for publication December 8, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Yang, J. Yang, Z. Yang, P. Chen, A. Fraser, W. Zhang, H. Pang, X. Gao, B. Wilson, J.-S. Hong, et al. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 and PACAP4-6 Are Neuroprotective through Inhibition of NADPH Oxidase: Potent Regulators of Microglia-Mediated Oxidative Stress J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 595 - 603. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
