Ii

H*YK

R

tgAidclioaPwPnrsgm

s

mmpnptpb

enddwPs

Biochemical and Biophysical Research Communications 271, 620–625 (2000)

doi:10.1006/bbrc.2000.2679, available online at http://www.idealibrary.com on

0CA

ncrease in Phospholipase C-d1 Protein Levelsn Aluminum-Treated Rat Brains

iroko Tanino,* Shun Shimohama,† Yoshinori Sasaki,* Yasuo Sumida,* and Sadaki Fujimoto*Department of Environmental Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cyo, Misasagi,amashinaku, Kyoto 607-8414, Japan; and †Department of Neurology, Faculty of Medicine,yoto University, 54 Shogoin-Kawaharacyo, Sakyoku, Kyoto 606-8507, Japan

eceived April 16, 2000

zyme in the phosphoinositide signal transductionmi(sb(stf

ecdlsaholpanmmbtb

w(mAPebeasg

The effect of administration of aluminum to rats onhe level of three phospholipase C (PLC) isozymes (b1,1, and d1) was assessed in a variety of brain tissues.fter exposure to aluminum, a statistically significant

ncrease in malondialdehyde, an index of lipid peroxi-ation, was observed. In addition, there was a signifi-ant reduction in the catalytic activity of low molecu-ar weight phosphotyrosine phosphatase, which losests activity during oxidative stress. This suggests thatxidative stress is induced in brain tissues exposed toluminum. The protein level of PLC-d1, but not that ofLC-b1 or -g1, was significantly increased in brainshere oxidative stress had been induced. The totalLC activity in aluminum-treated rat brains was sig-ificantly higher than that in control brains. Theseesults suggest that PLC-d1 protein levels in brain tis-ues are increased by the induction of oxidative stress,iving an explanation for its up-regulation in Alzhei-er’s disease. © 2000 Academic Press

Key Words: phospholipase C; aluminum; oxidativetress; brain; Alzheimer’s disease.

Extracellular signals are transmitted across the cellembrane by a variety of mechanisms that use secondessenger molecules. Phosphoinositide-specific phos-

holipase C (PLC) catalyzes the hydrolysis of phosphoi-ositides to generate diacylglycerol and three inositolhosphates, of which diacylglycerol and inositol 1,4,5-risphosphate, serve as intracellular messengers forrotein kinase C activation and intracellular Ca21 mo-ilization, respectively (1–4). Thus, PLC is a key en-

Abbreviations used: PLC, phospholipase C; AD, Alzheimer’s dis-ase; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PMSF, phe-ylmethylsulfonyl fluoride; DFP, diisopropylfluorophosphate; DTT,ithiothreitol; EGTA, ethylene glycol tetraacetic acid; MDA, malon-ialdehyde; TBA, thiobarbituric acid; LMW-PTP, low moleculareight phosphotyrosine phosphatase; SDS, sodium dodecyl sulfate;AGE, polyacrylamide gel electrophoresis; PBS, phosphate-bufferedaline.

620006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

echanism. A comparison of amino acid sequences hasndicated that PLC can be divided into three typesPLC-b, -g, and -d) and that each type includes multipleubtypes. To date, more than 10 PLC isozymes haveeen identified by protein chemistry or cDNA cloning5). It is now recognized that the PLC-b family is as-ociated with G proteins, and the PLC-g family withyrosine kinases. However, little is known about theunction of the PLC-d family.

Alzheimer’s disease (AD) is a neurodegenerative dis-ase characterized by the progressive deterioration ofognitive function and memory (6). We have previouslyemonstrated that a PLC isozyme, PLC-d1, accumu-ates abnormally in neurofibrillary tangles, neuritesurrounding senile plaque cores, and neuropil threads,ll pathological characteristics of AD (7). Moreover, weave shown, using gel filtration chromatography, thatf the PLC isozymes, PLC-d1 activity and its proteinevels are significantly increased in AD brains com-ared with controls (8). The mechanism of increasedccumulation of PLC-d1 in the AD brain, however, hasot been clarified. Thus, studies to determine theechanism of increased PLC-d1 protein in AD brainsight be important in clarifying the close association

etween alterations in phospholipid-specific signalransduction and key features of AD such as neurofi-rillary degeneration and neuronal death.Recently, a cDNA corresponding to a putative PLCas cloned from the higher plant Arabidopsis thaliana

At). In a comparison with PLC-b, -g, and -d fromammalian cells, the overall structure of this putativet-PLC protein was found to be most similar to that ofLC-d. Moreover, it was shown that the At-PLC gene isxpressed at very low levels under normal conditionsut is induced to significantly higher levels by variousnvironmental stresses, such as dehydration, salinity,nd low temperature (9). We have previously demon-trated that exposure of cultured cortical neurons tolutamate causes neuronal death and an increase in

PoNl(tpoP

lwidwa(

M

t[R(aCa

rtGdraCpcib

bw1cT

7.0) containing 0.32 M sucrose, 0.1 mM PMSF, 0.5 mM DFP, 1 mMdmt1thcb

dc(uiree

luaaoo

s(LpMbdna11hnHmrJoooaa

TABLE 1

CA

idts

HCC

irat

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LC-d1 immunoreactivity and suggested that nitricxide (NO) formation, secondary to Ca21 influx after-methyl-D-aspartate (NMDA) receptor activation,

eads to changes in PLC-d1 similar to those seen in AD10). In addition, there is evidence for the hypothesishat oxidative stress is closely associated with theathogenesis of AD (11). These findings suggest thatxidative stress in the brain causes the induction ofLC-d1.In the present study we examined alterations in the

evels of PLC isozymes in various regions of rat brainshich had been subjected to oxidative stress. Admin-

stration of aluminum was the method selected to in-uce oxidative stress because of previous findings inhich in vivo administration of aluminum resulted inn increase in lipid peroxidation levels in brain tissues12, 13).

ATERIALS AND METHODS

Materials. Aluminum lactate and 2-thiobarbituric acid were ob-ained from Wako Chemicals (Osaka, Japan). L-a-phosphatidylmyo-inositol 2-3H(N)] (1.0 Ci/mmol) was purchased from Americanadiolabeled Chemicals (St. Louis, MO). L-a-phosphatidylinositol

soybean, ammonium salt), phenylmethylsulfonyl fluoride (PMSF),nd diisopropylfluorophosphate (DFP) were obtained from Sigmahemical (St. Louis, MO). All other chemicals were of reagent gradend were obtained commercially.

Animals and experimental design. Three-week-old male Wistarats were purchased from Japan SLC (Kyoto). The animals werereated in accordance with the guidelines published in the NIHuide for the Care and Use of Laboratory Animals. Rats were ran-omly divided into the two groups. Aluminum-treated groups wereeceived aluminum lactate dissolved in 0.9% NaCl at a dose of 10 mgluminum/kg body weight intraperitoneally, for a period of 4 weeks.ontrol groups were received an equal volume of 0.9% NaCl intra-eritoneally, for the same period. Rats were killed after 4 weeks ofontinuous treatment and the brains were removed and dissectednto the following regions: cerebral cortex, hippocampus, and cere-ellum.

Preparation of brain extracts. For the lipid peroxidation assay,rain tissue samples (cerebral cortex, hippocampus, and cerebellum)ere homogenized with a Teflon-glass homogenizer in 4 volumes of.15% KCl. For the other experiments, brain tissue samples (cerebralortex, hippocampus, and cerebellum) were homogenized with aeflon-glass homogenizer in 4 volumes of 10 mM Hepes buffer (pH

Effect of Aluminum Administration on the Bodyand Brain Weight of Rats

Initial bodyweight (g)

Final bodyweight (g)

Brainweight (g)

ontrol group 48.9 6 0.8 219 6 2.9 1.68 6 0.02luminum-treated group 48.6 6 0.9 161 6 7.5* 1.56 6 0.03*

Note. Rats were treated with 10 mg aluminum/kg body weightntraperitoneally for 4 weeks and their body weight as recordeduring the course of treatment. The brain weight was determined athe end of treatment. Values are mean 6 SE (n 5 10). *P , 0.01,tatistically significant from control group.

621

ithiothreitol (DTT), 10 mg/mL aprotinin, 5 mg/mL pepstatin A, 5g/mL leupeptin, 5 mM benzamidine, and 4 mM ethylene glycoletraacetic acid (EGTA). The homogenate was centrifuged at05,000g for 60 min and the supernatant thus obtained was used ashe cytosolic fraction. The pellet was washed twice, suspended inomogenization buffer, and used as the particulate fraction. Proteinoncentration was determined by the method of Bradford (14) withovine serum albumin as the standard.

Malondialdehyde (MDA) determination. Lipid peroxidation wasetermined by measuring MDA, an index of lipid peroxidation, ac-ording to the method of Kikugawa et al. (15). Butylhydroxy toluen0.01%) was introduced into each assay mixture in order to preventndesirable autoxidation of the sample during the assay. The MDA

n the samples was reacted with thiobarbituric acid (TBA) and theesulting chromophore was detected at 532 nm. The results werexpressed as nmoles of MDA per mg of protein using the molarxtinction coefficient of the MDA-TBA chromophore (1.56 3 105).

Antibodies. Specific antibodies against PLC-d1 and low molecu-ar weight phosphotyrosine phosphatase (LMW-PTP) were preparedsing PLC-d1 protein produced by an E. coli expression system (16)nd bovine brain LMW-PTP (17), respectively. The characterizationnd specificity of these antibodies have been fully described previ-usly (7, 17). Specific antibodies against PLC-b1 and PLC-g1 werebtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunochemical detection. Proteins in the particulate and cyto-olic fractions from rat brains, dissolved in Laemmli sample buffer18), were subjected to 4–20% (for PLC isozymes) or 15–25% (forMW-PTP) sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-horesis (PAGE), and blotted onto Immobilon (Millipore, Bedford,A). The Immobilon membrane was incubated with phosphate-

uffered saline (PBS) containing 0.1% Tween 20 (TPBS) and 5%ehydrated skim milk (Difco Laboratories, Detroit, MI) to blockonspecific protein binding. The membrane was then incubated withnti-PLC-b1 (1:500), -g1 (1:500), -d1 (1:5,000), and -LMW-PTP (1:0000) antibodies in TPBS containing 5% dehydrated skim milk for8 h at 4°C. Blots were then washed with TPBS, and incubated withorseradish peroxidase (HRP)-linked antibody against rabbit immu-oglobulin (Ig) (diluted 1:2000). Subsequently, membrane-boundRP-labeled antibodies were detected using the enhanced chemilu-inescence detection system (ECL kit, Amersham). Protein bands

eacting with antibodies were detected on radiographic film (X-OmatB-1, Kodak) 5 to 60 s after exposure. The integrated optical densityf the 150 kDa protein band recognized by the anti-PLC-b1 antibody,f the 145 kDa protein band recognized by the anti-PLC-g1 antibody,f the 85 kDa protein band recognized by the anti-PLC-d1 antibody,nd of the 20 kDa protein band recognized by the anti-LMW-PTPntibody were measured by a scanning densitometer (Arcus II, Agfa,

TABLE 2

Effect of Aluminum Administration on the Level of LipidPeroxidation in Different Regions of the Rat Brain

Control Aluminum-treated

(nmol MDA/mg of protein)

ippocampus 1.13 6 0.17 2.53 6 0.63*erebral cortex 6.41 6 0.29 9.62 6 0.29*erebellum 1.21 6 0.09 1.78 6 0.15*

Note. Rats were treated with 10 mg aluminum/kg body weightntraperitoneally for 4 weeks and lipid peroxidation in differentegions of the rat brain was assayed as described under Materialsnd Methods. Values are mean 6 SE (n 5 10). *P , 0.03, statis-ically significant from control group.

Gt

pameparam

R

E

twc

L

dt

rats are summarized in Table 2. After 4 weeks of treat-mopt

E

tPdostocaPiLainbtlb

D

icoppwlpw

pldp

TABLE 3

HCC

irat

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ermany); these were taken as an indication of the relative quanti-ies of PLC-b1, PLC-g1, PLC-d1, and LMW-PTP, respectively.

Enzyme assay. The activity of PLC was assayed using L-a-hosphatidylinositol and L-a-phosphatidyl [myo-inositol 2-3H(N)] assubstrate as described previously (19). LMW-PTP activity waseasured using p-nitrophenyl phosphate as a substrate in the pres-

nce of L-(1)-tartrate, which is a strong high molecular weight acidhosphatase inhibitor, as described previously (20). One unit of PLCnd LMW-PTP were defined as that amount of each enzyme whicheleased 1.0 nmol of myo-inositol from L-a-phosphatidylinositolnd p-nitrophenol from p-nitrophenyl phosphate, respectively, perinute.

ESULTS

ffect of Aluminum Administration on Body andBrain Weights of Rats

All rats survived the treatment period, but a statis-ically significant reduction in both brain and bodyeights was observed in the aluminum-treated group

ompared with the control group (Table 1).

ipid Peroxidation in Aluminum-Treated Rat Brains

The effects of aluminum treatment on lipid peroxi-ation, as measured by the formation of free MDA inhe cerebral cortex, hippocampus, and cerebellum, of

FIG. 1. Expression of PLC-b1, -g1, and -d1 in the cytosolic and paarticulate (P) fractions from the hippocampus (Hip), cerebral cortex (oaded onto the gel (10 mg protein/lane). Preparation of the cytosoliescribed under Materials and Methods. Specific antibodies immunorotein bands, respectively, on SDS–PAGE as indicated by arrows.

Effect of Aluminum Administration on LMW-PTP Activityin Different Regions of the Rat Brain

Control Aluminum-treated

(nmol/min/mg of protein)

ippocampus 35.1 6 1.8 31.2 6 1.6*erebral cortex 43.3 6 1.6 39.9 6 1.2*erebellum 64.0 6 1.2 58.7 6 1.5*

Note. Rats were treated with 10 mg aluminum/kg body weightntraperitoneally for 4 weeks and LMW-PTP activity in differentegions of the rat brain was assayed as described under Materialsnd Methods. Values are mean 6 SE (n 5 10). *P , 0.05, statis-ically significant from control group.

622

ent, a statistically significant increase in MDA wasbserved in all regions of the rat brain examined. Inarticular, the increase in the hippocampus was higherhan that in other regions of the brain.

ffect of Aluminum Administration on LMW-PTPin the Rat Brain

The effect of aluminum treatment on LMW-PTP ac-ivity in rat brain tissues was also determined. LMW-TP is a cytosolic enzyme whose catalytic activity isependent on a specific cysteine residue (21). Previ-usly, it has been shown that oxidative-stress-inducingystems, such as the thiol-group oxidant diamide (22),he vanadate/H2O2 system (23) and the autoxidizationf ascorbic acid in the presence of sub-micromolar con-entrations of iron (24), inhibited LMW-PTP activity innumber of cell lines. Therefore, we determined LMW-TP activity as an additional oxidative stress marker

n the brain tissues of rats exposed to aluminum.MW-PTP activity in the brains of rats treated withluminum for 4 weeks was significantly less than thatn the control rat brains (Table 3). Conversely, immu-oquantification of the 20 kDa protein band recognizedy the anti-LMW-PTP antibody in the cytosolic frac-ion showed that there was no difference in the proteinevels of LMW-PTP in aluminum-treated and controlrains (data not shown).

etection of PLC Isozymes in Rat Brain Tissues

PLC isozymes in rat brain tissues were detectedmmunochemically by immunoblotting with the spe-ific antibodies. PLC-b1, -g1, and -d1 were recognizedn SDS–PAGE as 150 kDa-, 145 kDa-, and 85 kDa-rotein bands, respectively. Both PLC-g1 and -d1 wereresent mainly in the cytosolic fraction, while PLC-b1as also expressed at high levels in the particu-

ate fraction of the rat cerebral cortex, hippocam-us, and cerebellum. In addition, both PLC-b1 and -d1ere highly expressed in the hippocampus and cere-

ulate fractions of different regions of the rat brain. Cytosolic (C) and), and cerebellum (Cb) of seven-week-old male Wistar rat brains wered particulate fractions and immunoblot assays were performed as

emically recognized PLC-b1, -g1, and -d1 as 150-, 145-, and 85-kDa

rticCxc anch

bral cortex and at lower levels in the cerebellum, whilePo(

E

kbtpapiwtPcrbivtoa

E

atpa3cp8(au4etablatpt

D

p

isofissslcasazwta

atp(msPmc

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LC-g1 was expressed at almost the same level in eachf the hippocampus, cerebral cortex and cerebellumFig. 1).

ffect of Aluminum Administration on PLC ProteinLevels

Immunoquantification was performed on the 150Da-, 145 kDa-, and 85 kDa-protein bands recognizedy the anti-PLC-b1, -g1, and -d1 antibodies, respec-ively, in both the cytosolic and particulate fractionsrepared from the brain tissues of aluminum-treatednd control rats. Immunoquantification of the 85 kDarotein band recognized by the anti-PLC-d1 antibodyn the cytosolic fractions showed that the protein levelsere significantly higher in all of the aluminum-

reated brain tissues than in the control brains.LC-d1 protein levels in the particulate fraction of theerebral cortex and cerebellum from aluminum-treatedats were also significantly higher than in the controlrains, and those in the hippocampus also tended toncrease after aluminum-treatment (Fig. 2A). Con-ersely, there was no statistical difference in the pro-ein levels of PLC-b1 and -g1 in either the cytosolicr particulate fractions of rat brain tissues fromluminum-treated and control rats (Figs. 2B and 2C).

ffect of Aluminum Administration on PLC Activityin the Rat Brain

PLC activity in brain tissues from aluminum-treatednd control rats was determined. The PLC activity inhe cytosolic fraction of the cerebral cortex, hippocam-us, and cerebellum was significantly higher in theluminum-treated rats than that in control rats (Fig.): 76.8 6 4.8 units/mg protein (mean 6 SE) for controlerebral cortex (n 5 5) versus 105 6 8.4 units/mgrotein for aluminum-treated cerebral cortex (n 5 5);1.7 6 6.7 units/mg protein for control hippocampusn 5 5) versus 116 6 6.7 units/mg protein forluminum-treated hippocampus (n 5 5); 34.4 6 2.5nits/mg protein for control cerebellum (n 5 5) versus8.5 6 4.8 units/mg protein for aluminum-treated cer-bellum (n 5 5). PLC activity in the particulate frac-ion of the cerebellum from aluminum-treated rats waslso significantly higher than that in the control cere-ellum: 8.0 6 0.6 units/mg protein for control cerebel-um (n 5 5) versus 10.2 6 0.4 units/mg protein forluminum-treated cerebellum (n 5 5). PLC activity inhe particulate fraction of the cerebral cortex and hip-ocampus was not significantly different in aluminum-reated and control brains.

ISCUSSION

In the present study we examined alterations in therotein levels and activity of PLC isozymes (b1, g1, d1)

623

n rat brain tissues which were subjected to oxidativetress. We selected in vivo aluminum exposure as anxidative stress-inducing system, based on previousndings that in vivo administration of aluminum re-ults in an increase in lipid peroxidation in brain tis-ues (11, 12, 25). Under our experimental conditions atatistically significant increase in MDA, an index ofipid peroxidation, was detected in each of the cerebralortex, hippocampus, and cerebellum (Table 2). Welso determined LMW-PTP activity, another oxidativetress marker, in the brain tissues of control andluminum-treated rats. LMW-PTP is a cytosolic en-yme, previously known as LMW acid phosphatase,hose enzyme activity is dependent on a specific cys-

eine residue (21). Therefore, the enzyme loses its cat-lytic activity during oxidative stress (22–24). A signif-

FIG. 2. Effect of aluminum administration on PLC-b1, -g1,nd -d1 protein levels in different regions of the rat brain. Prepara-ion of the cytosolic (C) and particulate (P) fractions of the hippocam-us (Hip), cerebral cortex (Cx), and cerebellum (Cb) from controlopen bars) and aluminum-treated (closed bars) rat brains and im-unochemical detection of PLC isozymes were performed as de-

cribed under Materials and Methods. (A) PLC-d1 protein levels. (B)LC-b1 protein levels. (C) PLC-g1 protein levels. Bars indicateean 6 SE (n 5 10). *P , 0.05, statistically significant from

ontrol group.

ibicio

PissagbprrPrvi

dAptfsldvatma

influx by N-methyl-D-aspartate (NMDA) receptor acti-vPvscduslfimotdoi

ptm

A

fpFsS

R

dp(tpmg

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cant reduction in LMW-PTP activity was found in therain tissues of aluminum-treated rats (Table 3), whilets protein level was not altered. These findings indi-ate that the reduction of LMW-PTP activity is due tonactivation of the enzyme, confirming the occurrencef oxidative stress in aluminum-treated rat brains.The present study first demonstrated that of the

LC isozymes, protein levels of PLC-d1 are specificallyncreased in rat brain tissues subjected to oxidativetress by aluminum exposure (Fig. 2). In addition, atatistically significant increase in PLC activity waslso observed in aluminum-treated rats (Fig. 3), sug-esting that the increased PLC activity observed in ratrains exposed to aluminum is due to an increase in itsrotein levels. The loss in body and brain weights ofats administered with aluminum could be due to theetarded development of rats. However, increasedLC-d1 protein levels observed in aluminum-treatedat brains could not be attributed to the retarded de-elopment of rats because PLC-d1 was expressed sim-larly in rat brains from 4 to 8 postnatal weeks (26).

Recently, a cDNA corresponding to a putative PLC,esignated At-PLC-1, was cloned from the higher plantrabidopsis thaliana (At). The overall structure of thisutative At-PLC-1 protein was found to be most similaro that of PLC-d when compared with PLC-b, -g, and -drom mammalian cells. Interestingly, it was demon-trated that the At-PLC-1 gene is expressed at very lowevels in the plant under normal conditions but is in-uced to significantly higher levels under various en-ironmental stresses such as dehydration, salinity,nd low temperature (9). We have previously shownhat exposure of cultured rat cortical neurons to gluta-ate causes an increase in PLC-d1 immunoreactivity

nd suggested that NO formation secondary to Ca21

FIG. 3. Effect of aluminum administration on PLC activity inifferent regions of the rat brain. Preparation of the cytosolic (C) andarticulate (P) fractions of the hippocampus (Hip), cerebral cortexCx), and cerebellum (Cb) from control (open bars) and aluminum-reated (closed bars) rat brains and the assay for PLC activity wereerformed as described under Materials and Methods. Bars indicateean 6 SE (n 5 5). *P , 0.05, statistically significant from control

roup.

624

ation, leads to an increase in the protein levels ofLC-d (10). These findings suggest that PLC-d1 is in-olved in the signal transduction pathway of oxidativetress and that an increase in PLC-d1 protein levelsontributes to the response of mammalian cells to oxi-ative stress in a manner similar to At-PLC-1 contrib-ting to the response of the plant to environmentaltress (9). Therefore, an increase in PLC-d1 proteinevels and the close association of PLC-d1 with neuro-brillary tangles and senile plaques in AD (7, 8, 17, 19)ight be due to the response of neurons subjected to

xidative stress. Further studies are necessary to de-ermine whether increased PLC-d1 protein levels areue to a reduction in its degradation or due to itsverexpression, and to clarify the biological function ofts up-regulation against oxidative stress.

In conclusion, the present study shows that PLC-d1rotein levels are increased in brain tissues subjectedo oxidative stress, and this is the first observation inammalian cells.

CKNOWLEDGMENTS

This work was supported by Grants-in-Aid for Scientific Researchrom the Ministry of Education, Science, Sports and Culture of Ja-an, and grants from the Ministry of Welfare of Japan, the Inamorioundation, the Smoking Research Foundation for Scientific Re-earch, and the Promotion and Mutual Aid Corporation for Privatechool of Japan.

EFERENCES

1. Berridge, M. J., and Irvine, R. F. (1984) Inositol trisphosphate, anovel second messenger in cellular signal transduction. Nature312, 315–321.

2. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Bross,T. E., Bansal, S., and Wilson, D. B. (1986) The metabolism ofphosphoinositide-derived messenger molecules. Science 234,1519–1526.

3. Nishizuka, Y. (1988) The molecular heterogeneity of proteinkinase C and its implications for cellular regulation. Nature 334,661–665.

4. Rhee S. G., Suh P. G., Ryu S. H., and Lee S. Y. (1989) Studies ofinositol phospholipid-specific phospholipase C. Science 244, 546–550.

5. Noh, D. Y., Shin, S. H., and Rhee, S. G. (1995) Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim. Bio-phys. Acta 1242, 99–114.

6. Katzman, R. (1986) Alzheimer’s disease. N. Engl. Med. 314,964–973.

7. Shimohama, S., Homma, S., Suenaga, T., Fujimoto, S., Tanigu-chi, T., Araki, W., Yamaoka, Y., Takenawa, T., and Kimura, J.(1991) Aberrant accumulation of phospholipase C-delta in Alz-heimer Brains. Am. J. Pathol. 139, 737–742.

8. Shimohama, S., Sasaki, Y., Fujimoto, S., Kamiya, S., Taniguchi,T., Takenawa, T., and Kimura, J. (1998) Phospholipase Cisozymes in the human brain and their changes in Alzheimer’sdisease. Neuroscience 82, 999–1007.

9. Hirayama, T., Ohto, C., Mizoguchi, T., and Shinozaki, K. (1995)A gene encoding a phosphatidyl-specific phospholipase C is in-

duced by dehydration and salt stress in Arabidopsis thaliana.

1

1

1

1

1

1

1

1

18. Laemmli, U. K. (1970) Cleavage of structural proteins during the

1

2

2

2

2

2

2

2

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Proc. Natl. Acad. Sci. USA 92, 3903–3907.0. Shimohama, S., Akaike, A., Tamura, Y., Matsushima, H., Kume,

T., Fujimoto, S., Takenawa, T., and Kimura, J. (1995) Guluta-mate-induced antigenic changes of phospholipase C-delta in cul-tured cortical neurons. J. Neurosci. Res. 41, 418–426.

1. Markesbery, W. R. (1997) Oxidative stress hypothesis in Alzhei-mer’s disease. Free Radic. Bio. Med. 23, 134–147.

2. Ohtawa, M., Seko, M., and Takayama, F. (1983) Effect of alumi-num ingestion on lipid peroxidation in rats. Chem. Pharm. Bull.31, 1415–1418.

3. Julka, D., and Gill, K. D. (1996) Altered calcium homeostasis: Apossible mechanisms of aluminium-induced neurotoxicity. Bio-chim. Biophys. Acta 1315, 47–54.

4. Bradford, M. M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 248–254.

5. Kikugawa, K., Kojima, T., Yamaki, S., and Kosugi, H. (1992)Interpretation of the thiobarbitulic acid reactivity of rat liverand brain homogenates in the presence of ferric ion and ethyl-enediaminetetraacetic acid. Anal. Biochem. 202, 249–255.

6. Homma, Y., Emori, Y., Shibasaki, F., Suzuki, K., and Takenawa,T. (1990) Isolation and characterization of a gamma-typephosphoinositide-specific phospholipase C (PLC-gamma 2). Bio-chem. J. 269, 13–18.

7. Shimohama, S., Fujimoto, S., Chachin, M., Taniguchi, T., Perry,G., Whitehouse, P. J., and Kimura, J. (1995) Alterations of lowmolecular weight acid phosphatase protein level in Alzheimer’sdisease. Brain Res. 699, 125–129.

625

assembly of the head of bacteriophage T4. Nature 227, 680–685.9. Shimohama, S., Fujimoto, S., Matsushima, H., Takenawa, T.,

Taniguchi, T., Perry, G., Whitehouse, P. J., and Kimura, J. (1995)Alteration of phospholipase C-delta protein level and specific activ-ity in Alzheimer’s disease. J. Neurochem. 64, 2629–2634.

0. Shimohama, S., Fujimoto, S., Taniguchi, T., and Kimura, J.(1993) Reduction of low-molecular-weight acid phosphatase ac-tivity in Alzheimer brains. Ann. Neurol. 33, 616–621.

1. Ramponi, G. (1994) The low Mr cytosolc phosphotyrosine proteinphosphatase. Adv. Prot. Phosphatases 8, 1–25.

2. Monteiro, H. P., Ivaschenko, Y., Fischer, R., and Stern, A. (1991)Inhibition of protein tyrosine phosphatase activity by diamide isreversed by epidermal growth factor in fibroblasts. FEBS Lett.295, 146–148.

3. Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) The insulino-mimetic agents H2O2 and vanadate stimulate protein tyrosinephosphorylation in intact cells. J. Biol. Chem. 265, 2896–2902.

4. Monteiro, H. P., Ivaschenko, Y., Fischer, R., and Stern, A. (1993)Ascorbic acid inhibits protein tyrosine phosphatases in NIH 3T3cells expressing human epidermal growth factor receptors. Int.J. Biochem. 25, 1859–1864.

5. Julka, D., Vasishta, R. K., and Gill, K. D. (1996) Distribution ofaluminum in different brain regions and body organs of rat. Biol.Trace Elem. Res. 52, 181–192.

6. Shimohama, S., Sumida, Y., Fujimoto, S., Matsuoka, Y., Tanigu-chi, T., Takenawa, T., and Kimura, J. (1998) Differential expres-sion of rat brain phospholipase C isozymes in development andaging. Biochem. Biophys. Res. Commun. 243, 210–216.