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Krev-1 protein is abundantly expressed in the rat spinal cord
et Biophysics Acta
ELSEVIER
Biochimica et Biophysics Acta 1243 (1995) 446-452
Kreu-1 protein is abundantly expressed in the rat spinal cord Osamu Urayama a,b,*, Takayuki Murakoshi
‘, Yoji Ikawa b
aDepartment of Laboratory Medicine, Akita University School of Medicine, Hondo I -I-l, Akita 010, Japan ’ Department of Biochemistry, Tokyo Medical and Dental lJnil;ersity School of Medicine, Tokyo, Japan ’ Department of Pharmacology, Tokyo Medical and Dental University School of Medicine, Tokyo, Japan Received 18 July 1994; accepted 19 October 1994
Abstract The Kreu-1 gene, which was originally identified as a dominantly functioning tumor suppressor gene inducing a flat revertant of a v-K-ras-transformed NIH 3T3 cell line, was abundantly expressed in the mammalian brain [Kitayama et al. (1989) Cell 56, 77-841. To investigate where Kreu-1 and its family ras proteins are distributed in the central nervous system, we isolated the membrane fractions from several regions of the brain and spinal cord of rats by subcellular fractionation and analyzed those proteins by immunoblot analysis with the specific monoclonal antibodies. Kreu-1 protein was detected at the highest level in the spinal cord among areas of the central nervous system which included cerebral cortex, cerebellum, hippocampus, and olfactory bulb. On the other hand, ras proteins were found at similar levels in these regions. Within the spinal cord, Kreu-1 and rus proteins were detected at a comparable level in the ventral and dorsal parts, while they were much less in the dorsal root ganglion than in the spinal cord. They showed the differential expression during early postnatal development: Kreu-1 protein increased and ras proteins were at relatively high levels. When Kreu-1 and rus proteins were examined in synaptosomes from the lumbar spinal cord of newborn rats, most of them were detected not in the synaptic vesicles but in the synaptic plasma membranes. KreLi-1 protein as well as ras proteins might be involved in neuronal functions in the spinal cord such as sensory processing and motor control. Keywords: Krev-l/rap
l/
sq
21 protein; Ras protein; Brain; Spinal cord; Subcellular
distribution;
Synaptosome
-
1. Introduction Kreu-1 gene was isolated from a human fibroblast cDNA expression library with revertant-inducing activity on a v-K-rus-transformed NIH 3T3 cell line [l]. The Kreu-1 gene product, which is a IV, 21000 protein with 184 amino acids, belongs to a rus p2l/ras p21-like small GTP-binding protein superfamily (reviewed in Refs. [2-41). It is identical to human rap 1A protein [s] or bovine smg p21A [6] and homologous to rap lB/smg 21B [7,8], rap 2A [s] or rap 2B [9] protein. There are strong similarities between Kreu-1 and H-rus proteins in the guanine nucleotide binding region, the putative effector-binding re-
Abbreviations: Kreti-1, Kirsten-ras-revertant 1; Na,K-ATPase, Na+,K+-stimulated ATPase; SDS, sodium dodecyl sulfate; STE, 0.32 M sucrose/l0 mM Tris-HCl (pH 7.5)/l mM EDTA; SPM, synaptic plasma membrane; SV, synaptic vesicle; DRG, dorsal root ganglion; P2, crude mitochondrial fraction * Corresponding author. Fax: + 81 188 362624. 0304.4165/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved .SsDlO304-4165(94)00174-X
gion, and the C-terminal acylation site that is known to be involved in membrane attachment. It has been demonstrated, however, that the N-terminal region within Krev-1 protein, especially the sequence of Glu-30 and Lys-31 just before the effector-binding region, is essential for its reversion-inducing activity of the transformed state by the chimeric Kreu-1-H-rus gene study [lo] and point mutation study [ll]. Krev-l/rap lA/smg 21A mRNA and protein have been found ubiquitously in various mammalian tissues [1,5,12-141, and seem to be especially abundant in the brain [I]. Within the central nervous system, however, precise and quantitative analysis of the regional distribution has not been done yet. The subcellular distribution of Krev-1 or rap 1 proteins which has been reported by several groups still appears controversial. According to immunochemical analysis with the anti-smg p21 serum [12], rap 1 proteins have been found abundantly in the cytoplasmic region of neuronal cell bodies and moderately in neuropils of the rat brain and recovered mainly in the
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synaptosomal and mitochondrial fractions after subcellular fractionation. Rap 1A protein has been detected in the plasma membrane fraction. of human neutrophiles [14], and it has recently been reported that rap 1 proteins are associated with the Golgi complex of some culture cell lines [15]. On the other hand, ras proto-oncogenes and proteins were also abundantly detected in mammalian brains [16-181 and the proteins were shown to be highly concentrated in synaptic -plasma membranes [19,20]. It is interesting to learn what kind of physiological role Kreu-1 protein or ras proteins plays in non-proliferative cells such as neurons. In this paper, as an initial step to determine the possible physiological functions of Kreu-1 and ras in the central nervous system, we studlied the regional distribution of these proteins in the rat brain and spinal cord by immunoblot analysis with specific monoclonal antibodies. The results showed that both proteins were expressed to a high degree in the spinal cord, which suggests that they are involved in sensory and motor functions of spinal neurons.
2. Materials
and methods
2.1. Materials Antibody T22 [13] was the generous gift of H. Shiku, Nagasaki University School of Medicine, Nagasaki, Japan. Antibody Ras 10 was purchased from Oncogene Science. A horseradish peroxidase conjugated goat anti-mouse immunoglobulin was obtained from Tago or Zymed Lab. Clear Blot Membrane-P hydrophobic membrane was purchased from Atto Co. All reagents were of reagent grade. 2.2. Membrane
preparation
Wistar rats were used as the source of the membrane fractions of tissues. Newborns less than 4 days old were obtained from timed pregnant rats. Rats were deeply anesthesized under ether. Several regions of the brain and spinal cord were quickly dissected and frozen in liquid nitrogen. All procedures of membrane preparation were carried out in an ice bath. When post-nuclear membranes were fractionated, the STE buffer consisting of 0.32 M sucrose, 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA was used as homogenizing medium. The minced tissue was added at a ratio of N 50 mg to 450 ~1 of the STE buffer and homogenized with 15 strokes of a motor-driven Potter-Elvehjem homogenizer using a Teflon pestle. The homogenate was loaded ton 500 ~1 of fresh STE buffer in a 1.5 ml microtube and centrifuged at 850 X g for 20 min. The supernatant was recentrifuged at 300000 X g for 10 min in a Beckman TL-100 centrifuge and the pellet (postnuclear membranes) was suspended in 0.25 M sucrose and stored quickly at -80°C. In some experiments the super-
447
natant following removal of mitochondria and myelin fragments (8500 X g, 20 min) was ultracentrifuged to obtain microsomal membranes. When synaptic plasma membranes were isolated from the fresh spinal cord of newborn rats, a solution of 0.32 M sucrose was used as homogenizing medium as described by Kurokawa et al. [21]. 800 mg of the lumbar spinal cord (Ll-L5) was removed from 20-25 newborn rats for a single preparation. The procedure was described previously [33]. To characterize synaptic plasma membrane fractions, assays of the ouabain-sensitive Na,K-ATPase activity (1 unit = pmol Pi/h per mg of protein) and specific antibody binding were carried out [22]. Protein concentration was determined by the Lowry method [23] with BSA as a standard. 2.3. Electrophoresis
and immunoblotting
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as described [24]. Two sets of antigen, membrane proteins of less than 20 pg, were dissolved in SDS with EDTA, phenylmethylsulfonyl fluoride and 2-mercaptoethanol, heated, applied to the Laemmli discontinuous gel system with a 12.5% separating gel, and electrophoresed. Proteins of one set were transferred to hydrophobic membrane and the blotted filter was incubated with a 1:500 diluted solution of the T22 monoclonal antibody specific to K reu-l/smg 21A protein or with a 1:200 diluted solution of the Ras 10 anti-Ras monoclonal antibody at 20°C for 60 min. The bound antibody was detected with a horseradish peroxidase conjugated goat anti-mouse immunoglobulin. Proteins of another set were stained with Coomassie brilliant blue. Immunoblots and Coomassie blue-stained gels were subjected to scanning with a densitometer (Bio-Rad Model 620 or Shimazu CS 9000). Independent control experiments in which dose of an antigen was varried over a range of concentrations showed that the optical density of the stained bands was proportional to protein concentration (up to 24 pg of membrane proteins for Kreu-1 protein or 16 pg of membrane proteins for ras protein). The peak area of the stained band was normalized using the total amount of proteins present in one lane and relative staining levels were compared among the samples examined. To estimate the relative molecular weights of Kreu-1 or ras proteins, BSA (M, 67000), carbonic anhydrase (M, 29 OOO), soybean trypsin inhibitor (M, 20 loo>, myoglobin (M, 17 000) and lysozyme (M, 14300) were used. A plot of the mobilities against the known molecular weights on a semilogarithmic scale gave a linear relationship in the range of M, 14 000-30 000. 2.4. Statistical analysis Data were expressed as mean + S.D., and P values were determined by Student’s unpaired t test.
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1
3. Results 3.1. Characterization
2
3
4
5 - 29k
of antibodies
A
Monoclonal antibody T22 was raised against the human smg 21A/Kreu-1 protein produced in Escherichia coli [13]. To confirm whether the antibody can recognize the rat Kreu-1 protein, we examined antibody binding to some subcellular fractions from rat brain tissue by immunoblot analysis. Antibody T22 detected only one protein band with M, 22000 not in the cytosol fraction but in the particulate fraction. The protein size was roughly similar to that described before [ 131. Anti-Ras monoclonal antibody, Ras 10, which can cross-react with H-, K- and N-ras proteins [25], gave two closely spaced bands (M, values 22 500 and 21000) in the particulate fraction of rat brain.
B
6
7
8
9
10 -
29k
3.2. Distribution of Krev-I protein in the brain and spinal cord
Kreu-1 mRNA has been found in abundance in rat brain 111. To assess the relative abundance of Kreu-1 protein and its family ras proteins in the rat central nervous system, post-nuclear membrane fractions, without potential selective loss of proteins by membrane purification, were prepared from several regions of the brain and spinal cord of 28 day-old rats, and analyzed by immunoblot analysis with Antibody T22 or Antibody Ras 10. Fig. 1 shows a representative immunoblot. Kreu-1 protein was detected at the highest level in the spinal cord and at the lowest level in the hippocampus among all regions examined (Fig. 1A). The levels of Kreu-1 proteins of the brain regions relative to that of the spinal cord (1.00) are estimated in Table 1: 0.64 5 0.26 for cerebral cortex (mean k S.D., n = 4, P < 0.05); 0.43 + 0.09 for hippocampus (P < 0.001); 0.66 f 0.11 for cerebellum (P < 0.01); 0.51 k 0.06 for olfactory bulb (P < 0.001). The levels of ras proteins, on the other hand, were relatively similar in all brain regions (Fig. 1C).
Fig. 1. Immunoblot analysis of Kreu-1 and ras proteins in rat brain and spinal cord. Spinal cord, cerebral cortex, hippocampus, cerebellum and olfactory bulb were dissected from 28 day-old rats and membranes were fractionated. Membrane proteins at the same amount of total protein among the regions were electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A) and Ras 10 (C) as described in Materials and Methods. 20 pg and 10 pg of the antigen protein were used for T22 and Ras 10, respectively. B and D show Coomassie blue-stained protein bands (30 pg applied) between M, values 30000 and 20000. The size of carbonic anhydrase (M, 29000) is marked with 29 K. Lanes 1 and 7 are spinal cord; lanes 2 and 6, cerebral cortex; lanes 3 and 8, hippocampus; lanes 4 and 9, cerebellum; lanes 5 and 10, olfactory bulb. The control without antibodies gave few staining bands.
3.3. Distribution within the spinal cord The relative abundance of Kreu-1 and ras proteins was compared along the rostro-caudal axis of the spinal cord as well as among ventral horn, dorsal horn, and dorsal root ganglia (DRGs). Primary sensory neurons convey sensory signals from the periphery to the spinal cord, with their somata located in the DRGs. In the dorsal horn of the spinal cord, central brancjes of the DRG neurons terminate and sensory information is integrated. Therefore, DRGs and the dorsal horn primarily participate in sensory functions while the ventral horn where motoneurons reside is basically concerned in motor functions. The thoracolumbar spinal cords are the site of sympathetic preganglionic neurons and parasympathetic preganglionic neurons locate in the sacral segments.
Table 1 Abundance
of Kreu-1
spinal cord cerebral cortex hippocampus cerebellum olfactory bulb
and ras proteins in brain and spinal cord Kreu-1
Ras
1 00 0.64 0.43 0.66 0.51
1.00 1.09 + 0.26 1.21*0.29 1.19L-0.46 0.89kO.18
a,b,c,d
a f 0.26 b,e + - 0 .09 c,e +0.11 d $06
The relative levels were calculated from the results presented in Fig. 1 as described in Materials and Methods. Mean+ S.D. is shown of four experiments and statistical comparisons were made using an unpaired t
test: a P < 0.05; bP cP dP eP
< < < <
0.001; 0.01; 0.001; 0.05.
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The spinal cords from 28 day-old rats were dissected into the cervical, thoracic, lumbar, sacral spinal cords and the DRGs of the entire spinal length. The lumbar spinal cord was further separated into two parts, dorsal and ventral halves. Post-nuclear membrane fractions were prepared from them and analyzed by immunoblot analysis in Fig. 2. Kreu-1 protein was detected at an equal level in the cervical, thoracic, lumbar and sacral spinal cords, but at a much lower level in the DRGs (Fig. 2A). Similar results were obtained for rus proteins (Fig. 2B). Both proteins were detected at equal levels in the ventral and dorsal parts (Fig. 2 D and E).
tive levels at developmental stages were as follows: 1.00 at 4 days; 1.27 f 0.37 (mean f S.D., n = 3) at 7 days; 1.70 h 0.34 at 14 days (P < 0.05 between 4 and 14 days); and 3.05 f 0.66 at 28 days (P < 0.01 between 4 and 28 days). In contrast, Antibody Ras 10 detected ras proteins during early development at a high level that did not change in the post-nuclear membrane fractions (Fig. 3C). In the microsomal fractions, however, ras proteins decreased slightly in the 28 day sample (Fig. 4B): 1.00 at 4 days; 1.12 & 0.23 (mean f S.D., n = 3) at 7 days; 0.82 f 0.05 at 14 days; 0.66 _t 0.09 at 28 days (P < 0.05 between 7 and 28 days).
3.4. Developmental
3.5. Distribution
change in the spinal cord
The expression of K rev-1 and ras proteins in the lumbar spinal cord of different ages (0, 4, 7 and 28 days) was examined by immunoblot analysis with post-nuclear membrane fractions in Fig. 3. Antibody T22 stained Kreu-1 proteins at all ages examined (arrow in Fig. 3A). Other staining bands on the blot for O-day, 4-day and 7-day samples were visible without the antibody, which suggested non-specific binding (Fig. 3B). Therefore, the expression of Kreu-1 protein was examined by immunoblot analysis with the microsomal fractions of different ages (4, 7, 14 and 28 days) after the removal of mitochondria and myelin fragments from ihe post-nuclear membrane fractions. Fig. 4A shows a representative immunoblot. Antibody T22 detected only Kreu-1 protein, which increased gradually with aging during early development. The rela-
in the synaptosomes
To see whether Kreu-1 or ras protein is expressed in neuronal cells, the P2 fraction containing synaptosomes was prepared from the lumbar spinal cord of 5-6 day-old rats and further fractionated by ultracentrifugation over a discontinuous density gradient of sucrose. The synaptic plasma membrane fraction 1 (SPMl, 0.6-0.8 M sucrose layer), fraction 2 (SPM2, 0.8-1.0 M sucrose layer) and the synaptic vesicle fraction (SV, 0.4 M sucrose layer) obtained were characterized using the ouabain-sensitive Na,K-ATPase enzyme, a marker enzyme of the plasma membrane. The specific activities of the Na,K-ATPase increased to 26.9 units in the SPM 1 and 52.5 units in the SPM 2 from 1.3 units in the P2. The Na,K-ATPase activity could hardly be detected in the SV. The purity of these membranes was also confirmed by immunoblot analysis
12345
6 -2Qk
A
v_
7 -2Qk
Dumim
,w.
B
P
E
Fig. 2. Immunoblct analysis of Kreu-1 and ras proteins within spinal cord. The spinal cord dissected from 28-day-old rats was separated to cervical, thoracic, lumbar, sacral cords, and DRGs belonging to the whole segments were collected together. Another lumbar spinal cord was further separated into ventral and dorsal parts. Membranes were fractionated. Membrane proteins at the same amount of total protein among the regions were electrophoresed in SDS on 12.5% polyacrylamide gels as described in the Fig. 1 legend and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A,D) and Ras 10 (B,E). C and F show Coomassie blue-stained protein bands between IV, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is cervical cord; 2, thoracic cord, 3, lumbar cord; 4, sacral cord; 5, DRG; 6, ventral part of lumbar cord; 7, dorsal part of lumbar cord. In A, degradation products of Kreu-1 proteins may be weakly stained in the lower molecular weight region.
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A
B
c-
C
D Fig. 3. Immunoblot analysis of Kreo-1 and ras proteins in the post-nuclear membrane fractions of lumbar spinal cords during development. Membranes were fractionated from lumbar spinal cords of different ages. Membrane proteins at the same amount of total protein among ages were electrophoresed in SDS on 12.5% polyacrylamide gels as described in the Fig. 1 legend and blotted onto hydrophobic membrane. Strips of the blot were stained as follows: A, with T22; B, without T22; C, with Ras 10. D shows Coomassie blue-stained protein bands between M, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is 0 day-old; lane 2, 4 day-old; lane 3, 7 day-old; and lane 4, 28 day-old. The position of Kreu-1 protein is marked with an arrow in A or B.
with the anti-serum against the pig kidney Na,K-ATPase protein (not shown). Fig. 5 shows the result. Binding of Antibody T22 to the SPM was lo-fold stronger than that to the SV: 1.0 in SPMl, 0.9 in SPM2 and 0.1 in SV when immunoblot was quantitated (Fig. 5A). Antibody Ras 10 also detected ras proteins not in the synaptic vesicle fraction but in the synaptic plasma membrane fraction: 1.0 in SPM and 0.2 in SV, when another preparation was analyzed (Fig. 5C).
4. Discussion The results of the present study indicate that both Kreu-l/smg 21A/rap 1A protein and ras proteins are distributed throughout the rat central nervous system. Kreu-1 protein was detected more in the spinal cord than in some regions of the brain (Fig. 1). The spinal cord must be one of the tissues with the highest exoression of Krev-1 I
~~~~~~~
~~
~~
~~
since mammalian brain has been considered to contain this mRNA in abundance [l]. On the other hand, the levels of ras proteins were similar in the several regions examined (Fig. 1). It has been reported that both brain and spinal cord express ras proteins strongly [17,18]. Our present result, however, was not consistent with that of Furth et al. [17] using immunohistochemical analysis in the point that the expression of ras proteins in the DRG was much lower than that in the spinal cord. Within the rat spinal cord, Kreu-1 and ras proteins were expressed with some similarity. (1) They were abundantly detected in all the cervical, thoracic, lumbar and sacral spinal cords (Fig. 2). (2) They were much more detected in the spinal cord containing the dorsal and ventral horns than in the DRG (Fig. 2). (3) The amounts of their expressions between the dorsal and ventral parts of the lumbar spinal cord were compatible (Fig. 2). 4) They were localized much more in the plasma membrane than in the synaptic vesicle of synaptosomes of lumbar spinal neurons (Fig. 5). But they differed in abundance during early postnatal development: K rev-l protein increased gradually with aging and ras proteins were expressed at high and constant levels (Figs. 3 and 4). The result for ras proteins is in agreement with the previous observations by Northern blot analysis of mouse brain [16] and by immunohistochemical analysis comparing between fetal and adult rat brains [17]. The localization of ras proteins in the plasma membrane of synaptosome has also been indicated by immunoblot analyses from other laboratory [19,20]. But sub-
1 A
2 “--ws
3
4 - 29k
-
B
Fig. 4. Immunoblot analysis of Kreu-1 and ras proteins in the microsoma1 fractions of lumbar spinal cords during development. Membranes were prepared as described in Materials and Methods. Membrane proteins at the same amount of total protein among ages (8-10 up) were electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A) and Ras 10 (Bl. C shows Coomassie blue-stained protein bands between M, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is 4 day-old; lane 2, 7 day-old; lane 3, 14 day-old; and lane 4, 28 day-old. The control without antibodies gave no stains.
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et Biophysics Acta 1243 (1995) 446-452
\ *.,
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C Fig. 5. Immunoblot analysis of Krec;-1 and ras proteins in synaptosome fractions from lumbar spinal cord. Membranes were isolated from the P2 fraction as described in Materials and Methods. Five ug of the protein was electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A), without T22 (B), and with Ras 10 (Cl, respectively. In A and B, lane 1 is P2 supernatant; lane 2, synafltic plasma membrane 2; lane 3, synaptic plasma membrane 1; and lane 4, synaptic vesicle. In C, lane 5 is P2; lane 6, synaptic plasma membrane; and lane 7, synaptic vesicle. The molecular weight size of carbonic anhydrase is marked with 29 k. Representative results are shown, respectively, of two experiments.
cellular distribution of rap 1 proteins (rap lA/Kreul/smg 21A, rap lB/smg 21B or both) remains controversial, since a couple of reports have suggested that these are bound to cellular membranes other than plasma membranes. Kim et al. [12] reported that rap 1 proteins were recovered in the synaptic vesicle and mitochondrial fractions as well as in the synaptic plasma membrane fraction from rat cerebrum, and Beranger et al. [15] have recently reported that rap 1 proteins are associated with the Golgi apparatus in several culture cell lines. The antibodies used in these studies were raised against rap lB/smg 21B protein as well as Kreu-l/rap lA/smg 21A protein. The contradictory result may be attributable to the presence of two proteins, rap 1A and rap 1B. Rap 1B protein, which is 95% identical to rap 1A [7,8], also contains the C-terminal Cys residue motif allawing its association with membranes [4]. It has recently been shown to be selectively phosphorylated by CaM kinase Gr, a neuronal Ca*+/calmodulin-dependent protein kinase [26]. Where the rap 1B protein is localized in synaptosomes should be elucidated, and this will require the development of spe-
451
cific antibodies, especially anti-rap 1B antibody. Immunochemical studies using the anti-rap 1A protein, anti-rap 1B protein and anti-rus protein antibodies may be informative under the same conditions in some tissues [13]. In proliferative cells, Kreu-1 protein may regulate cellular proliferation by antagonizing the growth-promting action of rus proteins [1,3]. What kind of physiological function do these proto-oncogene products have in nonproliferative cells such as neurons? Rus proteins have been found to be widely expressed in differentiated cells where they are likely to fulfill a certain specific function [2,3]. Rus proteins can promote nerve growth factor-induced survival and fiber outgrowth of pheochromocytoma PC12 cells [27-301. Borasio et al. [31] have recently reported the involvement of rus proteins in the signal transduction of neurotrophic factors in sensory neurons. Our data have suggested that both KreLj-1 and rus proteins are expressed in the dorsal part containing the dorsal horn where primary sensory neurons terminate as well as in the ventral part containing the ventral horn where motoneurons reside. The result for the distribution of these proteins is supported by that of the Kreu-1 and c-H-rus mRNAs (Urayama et al., unpublished observation). We have recently observed that the expression levels of Kreu-1 and c-H-rus genes in rat spinal neurons are increased by peripheral noxious stimulation [32]. Further analysis is necessary to determine the physiological functions of these proto-oncogenes in the central nervous system.
Acknowledgements We thank Dr. Hiroshi Shiku (Nagasaki University) for his generous gift of Antibody T22, Hiroko Hamasaki and Hidenori Suzuki (Tokyo Medical and Dental University) for membrane preparation, and Masanori Otsuka (Tokyo Medical and Dental University) and Shiro Uesugi (Akita University) for advice and comments. This work was supported by Grants-in-Aid for Scientific Research (04255101, 04454154) from the Ministry of Education, Science and Culture of the Japanese Government.
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ELSEVIER
Biochimica et Biophysics Acta 1243 (1995) 446-452
Kreu-1 protein is abundantly expressed in the rat spinal cord Osamu Urayama a,b,*, Takayuki Murakoshi
‘, Yoji Ikawa b
aDepartment of Laboratory Medicine, Akita University School of Medicine, Hondo I -I-l, Akita 010, Japan ’ Department of Biochemistry, Tokyo Medical and Dental lJnil;ersity School of Medicine, Tokyo, Japan ’ Department of Pharmacology, Tokyo Medical and Dental University School of Medicine, Tokyo, Japan Received 18 July 1994; accepted 19 October 1994
Abstract The Kreu-1 gene, which was originally identified as a dominantly functioning tumor suppressor gene inducing a flat revertant of a v-K-ras-transformed NIH 3T3 cell line, was abundantly expressed in the mammalian brain [Kitayama et al. (1989) Cell 56, 77-841. To investigate where Kreu-1 and its family ras proteins are distributed in the central nervous system, we isolated the membrane fractions from several regions of the brain and spinal cord of rats by subcellular fractionation and analyzed those proteins by immunoblot analysis with the specific monoclonal antibodies. Kreu-1 protein was detected at the highest level in the spinal cord among areas of the central nervous system which included cerebral cortex, cerebellum, hippocampus, and olfactory bulb. On the other hand, ras proteins were found at similar levels in these regions. Within the spinal cord, Kreu-1 and rus proteins were detected at a comparable level in the ventral and dorsal parts, while they were much less in the dorsal root ganglion than in the spinal cord. They showed the differential expression during early postnatal development: Kreu-1 protein increased and ras proteins were at relatively high levels. When Kreu-1 and rus proteins were examined in synaptosomes from the lumbar spinal cord of newborn rats, most of them were detected not in the synaptic vesicles but in the synaptic plasma membranes. KreLi-1 protein as well as ras proteins might be involved in neuronal functions in the spinal cord such as sensory processing and motor control. Keywords: Krev-l/rap
l/
sq
21 protein; Ras protein; Brain; Spinal cord; Subcellular
distribution;
Synaptosome
-
1. Introduction Kreu-1 gene was isolated from a human fibroblast cDNA expression library with revertant-inducing activity on a v-K-rus-transformed NIH 3T3 cell line [l]. The Kreu-1 gene product, which is a IV, 21000 protein with 184 amino acids, belongs to a rus p2l/ras p21-like small GTP-binding protein superfamily (reviewed in Refs. [2-41). It is identical to human rap 1A protein [s] or bovine smg p21A [6] and homologous to rap lB/smg 21B [7,8], rap 2A [s] or rap 2B [9] protein. There are strong similarities between Kreu-1 and H-rus proteins in the guanine nucleotide binding region, the putative effector-binding re-
Abbreviations: Kreti-1, Kirsten-ras-revertant 1; Na,K-ATPase, Na+,K+-stimulated ATPase; SDS, sodium dodecyl sulfate; STE, 0.32 M sucrose/l0 mM Tris-HCl (pH 7.5)/l mM EDTA; SPM, synaptic plasma membrane; SV, synaptic vesicle; DRG, dorsal root ganglion; P2, crude mitochondrial fraction * Corresponding author. Fax: + 81 188 362624. 0304.4165/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved .SsDlO304-4165(94)00174-X
gion, and the C-terminal acylation site that is known to be involved in membrane attachment. It has been demonstrated, however, that the N-terminal region within Krev-1 protein, especially the sequence of Glu-30 and Lys-31 just before the effector-binding region, is essential for its reversion-inducing activity of the transformed state by the chimeric Kreu-1-H-rus gene study [lo] and point mutation study [ll]. Krev-l/rap lA/smg 21A mRNA and protein have been found ubiquitously in various mammalian tissues [1,5,12-141, and seem to be especially abundant in the brain [I]. Within the central nervous system, however, precise and quantitative analysis of the regional distribution has not been done yet. The subcellular distribution of Krev-1 or rap 1 proteins which has been reported by several groups still appears controversial. According to immunochemical analysis with the anti-smg p21 serum [12], rap 1 proteins have been found abundantly in the cytoplasmic region of neuronal cell bodies and moderately in neuropils of the rat brain and recovered mainly in the
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synaptosomal and mitochondrial fractions after subcellular fractionation. Rap 1A protein has been detected in the plasma membrane fraction. of human neutrophiles [14], and it has recently been reported that rap 1 proteins are associated with the Golgi complex of some culture cell lines [15]. On the other hand, ras proto-oncogenes and proteins were also abundantly detected in mammalian brains [16-181 and the proteins were shown to be highly concentrated in synaptic -plasma membranes [19,20]. It is interesting to learn what kind of physiological role Kreu-1 protein or ras proteins plays in non-proliferative cells such as neurons. In this paper, as an initial step to determine the possible physiological functions of Kreu-1 and ras in the central nervous system, we studlied the regional distribution of these proteins in the rat brain and spinal cord by immunoblot analysis with specific monoclonal antibodies. The results showed that both proteins were expressed to a high degree in the spinal cord, which suggests that they are involved in sensory and motor functions of spinal neurons.
2. Materials
and methods
2.1. Materials Antibody T22 [13] was the generous gift of H. Shiku, Nagasaki University School of Medicine, Nagasaki, Japan. Antibody Ras 10 was purchased from Oncogene Science. A horseradish peroxidase conjugated goat anti-mouse immunoglobulin was obtained from Tago or Zymed Lab. Clear Blot Membrane-P hydrophobic membrane was purchased from Atto Co. All reagents were of reagent grade. 2.2. Membrane
preparation
Wistar rats were used as the source of the membrane fractions of tissues. Newborns less than 4 days old were obtained from timed pregnant rats. Rats were deeply anesthesized under ether. Several regions of the brain and spinal cord were quickly dissected and frozen in liquid nitrogen. All procedures of membrane preparation were carried out in an ice bath. When post-nuclear membranes were fractionated, the STE buffer consisting of 0.32 M sucrose, 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA was used as homogenizing medium. The minced tissue was added at a ratio of N 50 mg to 450 ~1 of the STE buffer and homogenized with 15 strokes of a motor-driven Potter-Elvehjem homogenizer using a Teflon pestle. The homogenate was loaded ton 500 ~1 of fresh STE buffer in a 1.5 ml microtube and centrifuged at 850 X g for 20 min. The supernatant was recentrifuged at 300000 X g for 10 min in a Beckman TL-100 centrifuge and the pellet (postnuclear membranes) was suspended in 0.25 M sucrose and stored quickly at -80°C. In some experiments the super-
447
natant following removal of mitochondria and myelin fragments (8500 X g, 20 min) was ultracentrifuged to obtain microsomal membranes. When synaptic plasma membranes were isolated from the fresh spinal cord of newborn rats, a solution of 0.32 M sucrose was used as homogenizing medium as described by Kurokawa et al. [21]. 800 mg of the lumbar spinal cord (Ll-L5) was removed from 20-25 newborn rats for a single preparation. The procedure was described previously [33]. To characterize synaptic plasma membrane fractions, assays of the ouabain-sensitive Na,K-ATPase activity (1 unit = pmol Pi/h per mg of protein) and specific antibody binding were carried out [22]. Protein concentration was determined by the Lowry method [23] with BSA as a standard. 2.3. Electrophoresis
and immunoblotting
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as described [24]. Two sets of antigen, membrane proteins of less than 20 pg, were dissolved in SDS with EDTA, phenylmethylsulfonyl fluoride and 2-mercaptoethanol, heated, applied to the Laemmli discontinuous gel system with a 12.5% separating gel, and electrophoresed. Proteins of one set were transferred to hydrophobic membrane and the blotted filter was incubated with a 1:500 diluted solution of the T22 monoclonal antibody specific to K reu-l/smg 21A protein or with a 1:200 diluted solution of the Ras 10 anti-Ras monoclonal antibody at 20°C for 60 min. The bound antibody was detected with a horseradish peroxidase conjugated goat anti-mouse immunoglobulin. Proteins of another set were stained with Coomassie brilliant blue. Immunoblots and Coomassie blue-stained gels were subjected to scanning with a densitometer (Bio-Rad Model 620 or Shimazu CS 9000). Independent control experiments in which dose of an antigen was varried over a range of concentrations showed that the optical density of the stained bands was proportional to protein concentration (up to 24 pg of membrane proteins for Kreu-1 protein or 16 pg of membrane proteins for ras protein). The peak area of the stained band was normalized using the total amount of proteins present in one lane and relative staining levels were compared among the samples examined. To estimate the relative molecular weights of Kreu-1 or ras proteins, BSA (M, 67000), carbonic anhydrase (M, 29 OOO), soybean trypsin inhibitor (M, 20 loo>, myoglobin (M, 17 000) and lysozyme (M, 14300) were used. A plot of the mobilities against the known molecular weights on a semilogarithmic scale gave a linear relationship in the range of M, 14 000-30 000. 2.4. Statistical analysis Data were expressed as mean + S.D., and P values were determined by Student’s unpaired t test.
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1
3. Results 3.1. Characterization
2
3
4
5 - 29k
of antibodies
A
Monoclonal antibody T22 was raised against the human smg 21A/Kreu-1 protein produced in Escherichia coli [13]. To confirm whether the antibody can recognize the rat Kreu-1 protein, we examined antibody binding to some subcellular fractions from rat brain tissue by immunoblot analysis. Antibody T22 detected only one protein band with M, 22000 not in the cytosol fraction but in the particulate fraction. The protein size was roughly similar to that described before [ 131. Anti-Ras monoclonal antibody, Ras 10, which can cross-react with H-, K- and N-ras proteins [25], gave two closely spaced bands (M, values 22 500 and 21000) in the particulate fraction of rat brain.
B
6
7
8
9
10 -
29k
3.2. Distribution of Krev-I protein in the brain and spinal cord
Kreu-1 mRNA has been found in abundance in rat brain 111. To assess the relative abundance of Kreu-1 protein and its family ras proteins in the rat central nervous system, post-nuclear membrane fractions, without potential selective loss of proteins by membrane purification, were prepared from several regions of the brain and spinal cord of 28 day-old rats, and analyzed by immunoblot analysis with Antibody T22 or Antibody Ras 10. Fig. 1 shows a representative immunoblot. Kreu-1 protein was detected at the highest level in the spinal cord and at the lowest level in the hippocampus among all regions examined (Fig. 1A). The levels of Kreu-1 proteins of the brain regions relative to that of the spinal cord (1.00) are estimated in Table 1: 0.64 5 0.26 for cerebral cortex (mean k S.D., n = 4, P < 0.05); 0.43 + 0.09 for hippocampus (P < 0.001); 0.66 f 0.11 for cerebellum (P < 0.01); 0.51 k 0.06 for olfactory bulb (P < 0.001). The levels of ras proteins, on the other hand, were relatively similar in all brain regions (Fig. 1C).
Fig. 1. Immunoblot analysis of Kreu-1 and ras proteins in rat brain and spinal cord. Spinal cord, cerebral cortex, hippocampus, cerebellum and olfactory bulb were dissected from 28 day-old rats and membranes were fractionated. Membrane proteins at the same amount of total protein among the regions were electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A) and Ras 10 (C) as described in Materials and Methods. 20 pg and 10 pg of the antigen protein were used for T22 and Ras 10, respectively. B and D show Coomassie blue-stained protein bands (30 pg applied) between M, values 30000 and 20000. The size of carbonic anhydrase (M, 29000) is marked with 29 K. Lanes 1 and 7 are spinal cord; lanes 2 and 6, cerebral cortex; lanes 3 and 8, hippocampus; lanes 4 and 9, cerebellum; lanes 5 and 10, olfactory bulb. The control without antibodies gave few staining bands.
3.3. Distribution within the spinal cord The relative abundance of Kreu-1 and ras proteins was compared along the rostro-caudal axis of the spinal cord as well as among ventral horn, dorsal horn, and dorsal root ganglia (DRGs). Primary sensory neurons convey sensory signals from the periphery to the spinal cord, with their somata located in the DRGs. In the dorsal horn of the spinal cord, central brancjes of the DRG neurons terminate and sensory information is integrated. Therefore, DRGs and the dorsal horn primarily participate in sensory functions while the ventral horn where motoneurons reside is basically concerned in motor functions. The thoracolumbar spinal cords are the site of sympathetic preganglionic neurons and parasympathetic preganglionic neurons locate in the sacral segments.
Table 1 Abundance
of Kreu-1
spinal cord cerebral cortex hippocampus cerebellum olfactory bulb
and ras proteins in brain and spinal cord Kreu-1
Ras
1 00 0.64 0.43 0.66 0.51
1.00 1.09 + 0.26 1.21*0.29 1.19L-0.46 0.89kO.18
a,b,c,d
a f 0.26 b,e + - 0 .09 c,e +0.11 d $06
The relative levels were calculated from the results presented in Fig. 1 as described in Materials and Methods. Mean+ S.D. is shown of four experiments and statistical comparisons were made using an unpaired t
test: a P < 0.05; bP cP dP eP
< < < <
0.001; 0.01; 0.001; 0.05.
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The spinal cords from 28 day-old rats were dissected into the cervical, thoracic, lumbar, sacral spinal cords and the DRGs of the entire spinal length. The lumbar spinal cord was further separated into two parts, dorsal and ventral halves. Post-nuclear membrane fractions were prepared from them and analyzed by immunoblot analysis in Fig. 2. Kreu-1 protein was detected at an equal level in the cervical, thoracic, lumbar and sacral spinal cords, but at a much lower level in the DRGs (Fig. 2A). Similar results were obtained for rus proteins (Fig. 2B). Both proteins were detected at equal levels in the ventral and dorsal parts (Fig. 2 D and E).
tive levels at developmental stages were as follows: 1.00 at 4 days; 1.27 f 0.37 (mean f S.D., n = 3) at 7 days; 1.70 h 0.34 at 14 days (P < 0.05 between 4 and 14 days); and 3.05 f 0.66 at 28 days (P < 0.01 between 4 and 28 days). In contrast, Antibody Ras 10 detected ras proteins during early development at a high level that did not change in the post-nuclear membrane fractions (Fig. 3C). In the microsomal fractions, however, ras proteins decreased slightly in the 28 day sample (Fig. 4B): 1.00 at 4 days; 1.12 & 0.23 (mean f S.D., n = 3) at 7 days; 0.82 f 0.05 at 14 days; 0.66 _t 0.09 at 28 days (P < 0.05 between 7 and 28 days).
3.4. Developmental
3.5. Distribution
change in the spinal cord
The expression of K rev-1 and ras proteins in the lumbar spinal cord of different ages (0, 4, 7 and 28 days) was examined by immunoblot analysis with post-nuclear membrane fractions in Fig. 3. Antibody T22 stained Kreu-1 proteins at all ages examined (arrow in Fig. 3A). Other staining bands on the blot for O-day, 4-day and 7-day samples were visible without the antibody, which suggested non-specific binding (Fig. 3B). Therefore, the expression of Kreu-1 protein was examined by immunoblot analysis with the microsomal fractions of different ages (4, 7, 14 and 28 days) after the removal of mitochondria and myelin fragments from ihe post-nuclear membrane fractions. Fig. 4A shows a representative immunoblot. Antibody T22 detected only Kreu-1 protein, which increased gradually with aging during early development. The rela-
in the synaptosomes
To see whether Kreu-1 or ras protein is expressed in neuronal cells, the P2 fraction containing synaptosomes was prepared from the lumbar spinal cord of 5-6 day-old rats and further fractionated by ultracentrifugation over a discontinuous density gradient of sucrose. The synaptic plasma membrane fraction 1 (SPMl, 0.6-0.8 M sucrose layer), fraction 2 (SPM2, 0.8-1.0 M sucrose layer) and the synaptic vesicle fraction (SV, 0.4 M sucrose layer) obtained were characterized using the ouabain-sensitive Na,K-ATPase enzyme, a marker enzyme of the plasma membrane. The specific activities of the Na,K-ATPase increased to 26.9 units in the SPM 1 and 52.5 units in the SPM 2 from 1.3 units in the P2. The Na,K-ATPase activity could hardly be detected in the SV. The purity of these membranes was also confirmed by immunoblot analysis
12345
6 -2Qk
A
v_
7 -2Qk
Dumim
,w.
B
P
E
Fig. 2. Immunoblct analysis of Kreu-1 and ras proteins within spinal cord. The spinal cord dissected from 28-day-old rats was separated to cervical, thoracic, lumbar, sacral cords, and DRGs belonging to the whole segments were collected together. Another lumbar spinal cord was further separated into ventral and dorsal parts. Membranes were fractionated. Membrane proteins at the same amount of total protein among the regions were electrophoresed in SDS on 12.5% polyacrylamide gels as described in the Fig. 1 legend and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A,D) and Ras 10 (B,E). C and F show Coomassie blue-stained protein bands between IV, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is cervical cord; 2, thoracic cord, 3, lumbar cord; 4, sacral cord; 5, DRG; 6, ventral part of lumbar cord; 7, dorsal part of lumbar cord. In A, degradation products of Kreu-1 proteins may be weakly stained in the lower molecular weight region.
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A
B
c-
C
D Fig. 3. Immunoblot analysis of Kreo-1 and ras proteins in the post-nuclear membrane fractions of lumbar spinal cords during development. Membranes were fractionated from lumbar spinal cords of different ages. Membrane proteins at the same amount of total protein among ages were electrophoresed in SDS on 12.5% polyacrylamide gels as described in the Fig. 1 legend and blotted onto hydrophobic membrane. Strips of the blot were stained as follows: A, with T22; B, without T22; C, with Ras 10. D shows Coomassie blue-stained protein bands between M, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is 0 day-old; lane 2, 4 day-old; lane 3, 7 day-old; and lane 4, 28 day-old. The position of Kreu-1 protein is marked with an arrow in A or B.
with the anti-serum against the pig kidney Na,K-ATPase protein (not shown). Fig. 5 shows the result. Binding of Antibody T22 to the SPM was lo-fold stronger than that to the SV: 1.0 in SPMl, 0.9 in SPM2 and 0.1 in SV when immunoblot was quantitated (Fig. 5A). Antibody Ras 10 also detected ras proteins not in the synaptic vesicle fraction but in the synaptic plasma membrane fraction: 1.0 in SPM and 0.2 in SV, when another preparation was analyzed (Fig. 5C).
4. Discussion The results of the present study indicate that both Kreu-l/smg 21A/rap 1A protein and ras proteins are distributed throughout the rat central nervous system. Kreu-1 protein was detected more in the spinal cord than in some regions of the brain (Fig. 1). The spinal cord must be one of the tissues with the highest exoression of Krev-1 I
~~~~~~~
~~
~~
~~
since mammalian brain has been considered to contain this mRNA in abundance [l]. On the other hand, the levels of ras proteins were similar in the several regions examined (Fig. 1). It has been reported that both brain and spinal cord express ras proteins strongly [17,18]. Our present result, however, was not consistent with that of Furth et al. [17] using immunohistochemical analysis in the point that the expression of ras proteins in the DRG was much lower than that in the spinal cord. Within the rat spinal cord, Kreu-1 and ras proteins were expressed with some similarity. (1) They were abundantly detected in all the cervical, thoracic, lumbar and sacral spinal cords (Fig. 2). (2) They were much more detected in the spinal cord containing the dorsal and ventral horns than in the DRG (Fig. 2). (3) The amounts of their expressions between the dorsal and ventral parts of the lumbar spinal cord were compatible (Fig. 2). 4) They were localized much more in the plasma membrane than in the synaptic vesicle of synaptosomes of lumbar spinal neurons (Fig. 5). But they differed in abundance during early postnatal development: K rev-l protein increased gradually with aging and ras proteins were expressed at high and constant levels (Figs. 3 and 4). The result for ras proteins is in agreement with the previous observations by Northern blot analysis of mouse brain [16] and by immunohistochemical analysis comparing between fetal and adult rat brains [17]. The localization of ras proteins in the plasma membrane of synaptosome has also been indicated by immunoblot analyses from other laboratory [19,20]. But sub-
1 A
2 “--ws
3
4 - 29k
-
B
Fig. 4. Immunoblot analysis of Kreu-1 and ras proteins in the microsoma1 fractions of lumbar spinal cords during development. Membranes were prepared as described in Materials and Methods. Membrane proteins at the same amount of total protein among ages (8-10 up) were electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A) and Ras 10 (Bl. C shows Coomassie blue-stained protein bands between M, values 30000 and 20000. The size of carbonic anhydrase is marked with 29 k. Lane 1 is 4 day-old; lane 2, 7 day-old; lane 3, 14 day-old; and lane 4, 28 day-old. The control without antibodies gave no stains.
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\ *.,
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C Fig. 5. Immunoblot analysis of Krec;-1 and ras proteins in synaptosome fractions from lumbar spinal cord. Membranes were isolated from the P2 fraction as described in Materials and Methods. Five ug of the protein was electrophoresed in SDS on 12.5% polyacrylamide gels and blotted onto hydrophobic membrane. Strips of the blot were stained with T22 (A), without T22 (B), and with Ras 10 (Cl, respectively. In A and B, lane 1 is P2 supernatant; lane 2, synafltic plasma membrane 2; lane 3, synaptic plasma membrane 1; and lane 4, synaptic vesicle. In C, lane 5 is P2; lane 6, synaptic plasma membrane; and lane 7, synaptic vesicle. The molecular weight size of carbonic anhydrase is marked with 29 k. Representative results are shown, respectively, of two experiments.
cellular distribution of rap 1 proteins (rap lA/Kreul/smg 21A, rap lB/smg 21B or both) remains controversial, since a couple of reports have suggested that these are bound to cellular membranes other than plasma membranes. Kim et al. [12] reported that rap 1 proteins were recovered in the synaptic vesicle and mitochondrial fractions as well as in the synaptic plasma membrane fraction from rat cerebrum, and Beranger et al. [15] have recently reported that rap 1 proteins are associated with the Golgi apparatus in several culture cell lines. The antibodies used in these studies were raised against rap lB/smg 21B protein as well as Kreu-l/rap lA/smg 21A protein. The contradictory result may be attributable to the presence of two proteins, rap 1A and rap 1B. Rap 1B protein, which is 95% identical to rap 1A [7,8], also contains the C-terminal Cys residue motif allawing its association with membranes [4]. It has recently been shown to be selectively phosphorylated by CaM kinase Gr, a neuronal Ca*+/calmodulin-dependent protein kinase [26]. Where the rap 1B protein is localized in synaptosomes should be elucidated, and this will require the development of spe-
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cific antibodies, especially anti-rap 1B antibody. Immunochemical studies using the anti-rap 1A protein, anti-rap 1B protein and anti-rus protein antibodies may be informative under the same conditions in some tissues [13]. In proliferative cells, Kreu-1 protein may regulate cellular proliferation by antagonizing the growth-promting action of rus proteins [1,3]. What kind of physiological function do these proto-oncogene products have in nonproliferative cells such as neurons? Rus proteins have been found to be widely expressed in differentiated cells where they are likely to fulfill a certain specific function [2,3]. Rus proteins can promote nerve growth factor-induced survival and fiber outgrowth of pheochromocytoma PC12 cells [27-301. Borasio et al. [31] have recently reported the involvement of rus proteins in the signal transduction of neurotrophic factors in sensory neurons. Our data have suggested that both KreLj-1 and rus proteins are expressed in the dorsal part containing the dorsal horn where primary sensory neurons terminate as well as in the ventral part containing the ventral horn where motoneurons reside. The result for the distribution of these proteins is supported by that of the Kreu-1 and c-H-rus mRNAs (Urayama et al., unpublished observation). We have recently observed that the expression levels of Kreu-1 and c-H-rus genes in rat spinal neurons are increased by peripheral noxious stimulation [32]. Further analysis is necessary to determine the physiological functions of these proto-oncogenes in the central nervous system.
Acknowledgements We thank Dr. Hiroshi Shiku (Nagasaki University) for his generous gift of Antibody T22, Hiroko Hamasaki and Hidenori Suzuki (Tokyo Medical and Dental University) for membrane preparation, and Masanori Otsuka (Tokyo Medical and Dental University) and Shiro Uesugi (Akita University) for advice and comments. This work was supported by Grants-in-Aid for Scientific Research (04255101, 04454154) from the Ministry of Education, Science and Culture of the Japanese Government.
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