Vol.
182,
No.
February
14,
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1992
1201-1207
~53 EXPRESSION IN BRAIN AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN THE RAT* Michael Chopp 1*3,Yi Li’, Zheng G. Zhang’,3, and Svend 0. Freytag Center for Stroke Research, Departments of ‘Neurology ,2Molecular Biology Hospital Health Science Center, Detroit, MI 48202
, Henry Ford
3Department of Physics , Oakland University, Rochester, MI 40309
Received
December
17,
1991
SUMMARY: The purpose of this study is to determine whether the p53 protein, a product of the ~53 tumor suppressor gene, that has been associated with the 72kDa heat shock protein (hsp72), is expressed in ischemic brain. Adult Wistar rats (n=5) were subjected to 120 minutes of middle cerebral artery occlusion. Twelve hours after reopening the artery, brain tissue was analyzed to determine the extent of neuronal damage (hematoxylin and eosin), and the distribution of ~53 and hsp72 (monoclonal antibodies). Our data demonstrate that ~53 is expressed in regions of neuronal necrosis; in contrast, morphologically intact neurons express hsp72 . The data suggest that the presence of ~53 is associated with cell death and that hsp72 may regulate p53 function. 0 1992 Academic Press, Inc.
The wild-type ~53 protein is the product of the ~53 tumor suppressor gene (1,2). Recent studies have demonstrated that the ~53 protein forms a complex with one or more of the major heat shock proteins (3,4). Heat shock proteins, particularly, brain after cerebral &hernia
(5,6).
the inducible hsp72, are found in
The role played by hsp72 in ischemic cell damage is
unknown, although there is evidence that hsp72 may protect tissue from a variety of insults (7,8). The ~53 protein has recently been shown to induce programmed cell death, apoptosis, in myeloid leukemic cells (9). Since hsp72 has been associated with ~53, and may regulate ~53, and hsp72 is strongly expressed in brain in response to ischemic injury, it is reasonable to test whether ~53 is, itself, expressed in cerebral tissue subjected to an ischemic insult.
*This work was supported in part from NINDS grant PO1 NS23393. Abbreviations: hsp, heat shock protein; MCA, middle cerebral artery.
1201
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182,
No.
3, 1992
BIOCHEMICAL
MATJBIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Five male Wistar rats weighing 260-300 grams were used. Middle cerebral artery (MCA) occlusion was induced for 120 min, using a method of intraluminal vascular occlusion (10). Rats were fasted overnight preceding surgery, but allowed water ad libidum. Halothane anesthesia (1% halothane, 69% N,O, 30% OJ was administered using a face mask. The femoral artery was cannulated for serial measurements of serum arterial pH, PO,, pC0,. Rectal temperature was maintained at 37°C using a feedback heating pad. The right common carotid artery, external carotid artery and internal carotid artery were isolated via a ventral midline incision. The distal end of the external carotid artery was ligated with 5-O silk suture at the branch of the occipital artery, and the origin of the external carotid artery was loosely tied with 5-O silk suture. Two microvascular clips were placed across the common carotid artery and the internal carotid artery. A 5-cm length of 4-O nylon monofilament with its tip rounded by heating near a flame, was introduced into the lumen of the external carotid artery through a puncture between the two silk sutures. The silk suture around the origin of the external carotid artery was tightened around the intraluminal nylon suture to prevent bleeding, and the two clips on the common carotid artery and the internal carotid artery were removed. Approximately 18.0-19.0 mm of the nylon suture, the length determined by body weight, was advanced into the internal carotid artery to block the origin of the MCA, and to enter the anterior cerebral artery. The skin incision was closed. Restoration of MCA blood flow was accomplished by withdrawing the intraluminal suture until the tip rested in the stump of the external carotid artery. Induction of ischemia was evident by slowing of the EEG. All rats with MCA occlusion exhibited focal neurologic deficits characterized by left hemiparesis with failure to extend the left forepaw. The avidin-biotin-peroxidase method previously described (11,12) was used to detect hsp72 and ~53. Twelve hours following recirculation, all rats were given an overdose of pentobarbital and fixed by transcardial perfusion with heparinized sodium phosphate buffer (pH 7.4), followed by 4% paraformaldehyde in the buffer. The brain was removed and placed in the same fixative overnight. Eight coronal sections of 3 mm thickness were obtained using a rodent brain matrix. Adjacent vibratome coronal sections (50 pm) were obtained, and reacted immunohistochemically with a mouse monoclonal antibody to hsp72, (C92, Amersham, PRN 1197, Cleveland OH), and with a mouse monoclonal antibody to ~53, (Ab-3, Oncogene Science, OP29, Uniondale, N.Y.). The hsp72 antibody was used in a 1:200 dilution, and incubated for 10 hours; the p53 antibody (20 pg/ml) was also incubated for 10 hours. Blocking of nonspecific background staining was accomplished with normal sheep whole serum. Endogenous peroxidase was blocked with H202 and methanol. Biotinylated sheep antimouse IgG was incubated for 8 hours. Sections were incubated with streptavidin “bridge” and biotinylated horseradish peroxidase for 1 hour each. Peroxidase was detected with diaminobenzidine. Sections were gelatin mounted on slides for light microscopic evaluation. Control sections were prepared on each animal by performing immunohistochemistry, except that primary antibody was deleted. The remaining coronal slices from these rat brains were embedded in paraffin. Sections (6 pm) adjacent to those cut on the vibratome were stained with hematoxylin and eosin (H&E) for neuronal evaluation. RESULTS All serum arterial values of pH, pQ, and pC0, were within normal ranges.
In all
animals, the hemisphere contralateral to that of the MCA occlusion did not exhibit obvious cell damage, or hsp72 and p53 induction. In the ipsilateral hemisphere to the MCA occlusion,a homogeneous distribution of neuronal necrosis (- 80%) was found in the caudate putamen, lateral globus pallidus, and olfactory tubercle and adjacent structures. 1202
Luminal
and focal
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
neuronal necrosis (- 50%) was observed in piriform, on H&E staining (Fig. la,b).
Morphologically
RESEARCH
COMMUNICATIONS
insular, and parietal cortices, identified
intact hsp72 positive neurons were present in
the limbic and frontal cortices (Fig. 2a,b), and the temporal and occipital cortices of the peripheral zone, and absent from the necrotic core. primarily
Diffuse ~53 staining was evident and
localized to areas of neuronal necrosis in the ischemic center (Fig. 3a,b).
animals, more intense p53 staining was noted in the cortex than in the striatum.
In all
The cellular
source of the p53 staining was not apparent.
~53 was either, not detected (3 animals), or
weakly detected (2 animals) in morphologically
intact neurons, that were positively stained for
hsp72, in areas peripheral and adjacent to the ischemic center. ~53 was not evident in the contralateral hemisphere.
DISCUSSION Our data indicate that ~53 is expressed in regions of severe ischemic cell damage twelve hours after transient focal cerebral &hernia.
Areas encompassing necrotic neurons exhibit
positive staining for ~53, while areas, in the ipsilateral lesioned hemisphere, with intact neurons, fail to exhibit detectable levels of ~53, or in the region adjacent to the ischemic center may weakly stain for ~53.
Neither ~53 or hsp72 are expressed in the contralateral hemisphere.
Thus, the presence of ~53 is associated with neuronal necrosis. In light of our observations that ~53 is present coincident with necrotic neurons, we speculate, that the induction of ~53 after ischemia may facilitate cell death. Induction of ~53 in injured tissue, which promotes cell death, may be the converse of the loss of ~53 in tumor, which promotes uncontrolled proliferation (13). We have not yet determined, via DNA fragmentation studies (14), whether apoptosis is the mechanism of neuronal death.
Our data also cannot distinguish whether ~53 is simply
secondary to cell death, or causes cell death. However, our novel finding of ~53 in ischemic brain tissue associated with cell death, lends credence to a role for ~53 in promoting ischemic cell damage. hsp72 is present in cerebral tissue where ~53 is absent or reduced. ~53 binds to heat shock proteins of the 70kDa family (3,4).
Since hsp72 is present in morphologically
intact
neurons, and absent in necrotic neurons, this suggests that hsp72 may be active in protecting tissue, either by complexing
to ~53, and thereby regulating ~53 expression at the post
translational level, or by altering the morphology of ~53, thereby affecting ~53 function and antibody recognition. In vitro studies have shown that ~53 binds to the cognate (4), and to the inducible heat shock proteins of the 70kDa family (3). In the present study, both ~53 and hsp72 have been measured in vivo in non-tumored cerebral tissue. We employed a monoclonal antibody to
1203
182,No.3,1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIO
Fig. Coronalsections(Qm) stainedwith H&E were obtainedfrom all animals12 hours after 2 hoursof transientMCA occlusion,asdescribedin the text. Histologicaldamagewas presentin the right hemisphere (R) asa homogeneous distributionof neuronalnecrosisin the caudateputamen,lateralglobuspallidus,andolfactorytubercleandadjacentstructures.Luminal and focal neuronalnecrosiswas observedin the piriform, insularand parietalcokes. b. Enlargedareaof partialcortex, from thebox outlinedin Fig. la; neuronalnecrosiswasevident in approximately50% of the neurons. a x 8 , b x 103. 1204
INS
Vol.
182, No. 3, 1992
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fie. hsp72immunoreactivityin vibratomesections(50pm)was obtainedfrom the same animalasin Fig. 1. Morphologicallyintacthsp72positiveneuronswerepresentin limbic and frontal cortex andabsentfrom the necroticcorein the right hemisphere (R). b. Enlargedarea from the box outlinedin Fig 2a; showshsp72positiveneurons. a x 8 , b x 103. 1205
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIf
3NS
Fig. 3a. ~53 immunoreactivityin vibratomesections(5Orm) was obtainedfrom the same animalasin Fig. 1. Diffusep53 stainingwasevidentin the areasof neuronalnecrosislocated in the ischemiccenter. In this animal ~53 was weakly stained in areas containing morphologicallyintact neuronsand hsp72positiveneurons b. Enlargedarea from the box outlinedin Fig. 3a showsdiffuse~53 staining.The cellularsourceof the ~53 stainingwasnot apparent. a x 8 , b x103.
1206
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
hsp72. This antibody has no detectable cross reactivity with the cognate heat shock 70kDa protein, and does not stain in the contralateral non-ischemic hemisphere.
Thus, our data is
consistent with an association of ~53 with hsp72. In summary, we have found, for the first time, ~53 in ischemic cerebral tissue. The ~53 gene has been linked with inhibition
of cell proliferation as well as death in neoplastic cells.
Our data suggest, that induction of p53 may play a similar role in promoting
cell death in
ischemic tissue, as in tumor. REFERENCES
1.
Dippold WG, Jay G, DeLeo AB, and Khoury G Old LJ (1981) Proc. Natl. Acad. Sci. USA 78: 1695-1699.
2.
Benchimol S, and Pim D Crawford L (1982) EMBO J. 1:1055-1062.
3.
Pinhasi-Kimhi 185.
4.
Hinds PW, Finlay CA, Frey AB, and Levine AJ (1987) Mol Cell Biol.
5.
Nowak TS Jr (1985) J Neurochem.
6.
Sharp FR, Lowenstein DL, Simon R, and Hisanaga K (1991) J Cereb Blood Flow Metab. 11:621-627.
7.
Barbe MF, Tytell M, Gower DJ, and Welch WJ (1988) Science. 241-1817-1820.
8.
Chopp M, Chen H, Ho K-L, Dereski MO, Brown E, Hetzel FW, and Welch KMA (1989) Neurology. 39: 1396-1398.
9.
Rouach YE Resnitzlq D, Lotem J, Sachs L, Kimchi A, and Oren M (1991) Nature. 3521345-347.
10.
Zea Longa E, Weinstein PR, Carlson S, and Cummins R (1989) Stroke. 20:84-91.
11.
Vass K, Welch WJ, and Nowak TS JR (1988) Acta Neuropathol.
12.
Chopp M, Li Y, Dereski MO, Levine SR, Yoshida Y, and Garcia JH (1991) Acta Neuropathol. In Press.
13.
Finlay CA Hinds PW, and Levine AJ (1989) Cell. 57:1083-1093.
14.
Wyllie AH (1980) Nature.
0, Michalovitz,
Ben-Zeev, A, and Gren Moshe (1986) Nature. 320: 182p. 2863-2869.
45:1635-1641.
284:555-556.
1207
7:128-135.
182,
No.
February
14,
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1992
1201-1207
~53 EXPRESSION IN BRAIN AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN THE RAT* Michael Chopp 1*3,Yi Li’, Zheng G. Zhang’,3, and Svend 0. Freytag Center for Stroke Research, Departments of ‘Neurology ,2Molecular Biology Hospital Health Science Center, Detroit, MI 48202
, Henry Ford
3Department of Physics , Oakland University, Rochester, MI 40309
Received
December
17,
1991
SUMMARY: The purpose of this study is to determine whether the p53 protein, a product of the ~53 tumor suppressor gene, that has been associated with the 72kDa heat shock protein (hsp72), is expressed in ischemic brain. Adult Wistar rats (n=5) were subjected to 120 minutes of middle cerebral artery occlusion. Twelve hours after reopening the artery, brain tissue was analyzed to determine the extent of neuronal damage (hematoxylin and eosin), and the distribution of ~53 and hsp72 (monoclonal antibodies). Our data demonstrate that ~53 is expressed in regions of neuronal necrosis; in contrast, morphologically intact neurons express hsp72 . The data suggest that the presence of ~53 is associated with cell death and that hsp72 may regulate p53 function. 0 1992 Academic Press, Inc.
The wild-type ~53 protein is the product of the ~53 tumor suppressor gene (1,2). Recent studies have demonstrated that the ~53 protein forms a complex with one or more of the major heat shock proteins (3,4). Heat shock proteins, particularly, brain after cerebral &hernia
(5,6).
the inducible hsp72, are found in
The role played by hsp72 in ischemic cell damage is
unknown, although there is evidence that hsp72 may protect tissue from a variety of insults (7,8). The ~53 protein has recently been shown to induce programmed cell death, apoptosis, in myeloid leukemic cells (9). Since hsp72 has been associated with ~53, and may regulate ~53, and hsp72 is strongly expressed in brain in response to ischemic injury, it is reasonable to test whether ~53 is, itself, expressed in cerebral tissue subjected to an ischemic insult.
*This work was supported in part from NINDS grant PO1 NS23393. Abbreviations: hsp, heat shock protein; MCA, middle cerebral artery.
1201
0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182,
No.
3, 1992
BIOCHEMICAL
MATJBIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Five male Wistar rats weighing 260-300 grams were used. Middle cerebral artery (MCA) occlusion was induced for 120 min, using a method of intraluminal vascular occlusion (10). Rats were fasted overnight preceding surgery, but allowed water ad libidum. Halothane anesthesia (1% halothane, 69% N,O, 30% OJ was administered using a face mask. The femoral artery was cannulated for serial measurements of serum arterial pH, PO,, pC0,. Rectal temperature was maintained at 37°C using a feedback heating pad. The right common carotid artery, external carotid artery and internal carotid artery were isolated via a ventral midline incision. The distal end of the external carotid artery was ligated with 5-O silk suture at the branch of the occipital artery, and the origin of the external carotid artery was loosely tied with 5-O silk suture. Two microvascular clips were placed across the common carotid artery and the internal carotid artery. A 5-cm length of 4-O nylon monofilament with its tip rounded by heating near a flame, was introduced into the lumen of the external carotid artery through a puncture between the two silk sutures. The silk suture around the origin of the external carotid artery was tightened around the intraluminal nylon suture to prevent bleeding, and the two clips on the common carotid artery and the internal carotid artery were removed. Approximately 18.0-19.0 mm of the nylon suture, the length determined by body weight, was advanced into the internal carotid artery to block the origin of the MCA, and to enter the anterior cerebral artery. The skin incision was closed. Restoration of MCA blood flow was accomplished by withdrawing the intraluminal suture until the tip rested in the stump of the external carotid artery. Induction of ischemia was evident by slowing of the EEG. All rats with MCA occlusion exhibited focal neurologic deficits characterized by left hemiparesis with failure to extend the left forepaw. The avidin-biotin-peroxidase method previously described (11,12) was used to detect hsp72 and ~53. Twelve hours following recirculation, all rats were given an overdose of pentobarbital and fixed by transcardial perfusion with heparinized sodium phosphate buffer (pH 7.4), followed by 4% paraformaldehyde in the buffer. The brain was removed and placed in the same fixative overnight. Eight coronal sections of 3 mm thickness were obtained using a rodent brain matrix. Adjacent vibratome coronal sections (50 pm) were obtained, and reacted immunohistochemically with a mouse monoclonal antibody to hsp72, (C92, Amersham, PRN 1197, Cleveland OH), and with a mouse monoclonal antibody to ~53, (Ab-3, Oncogene Science, OP29, Uniondale, N.Y.). The hsp72 antibody was used in a 1:200 dilution, and incubated for 10 hours; the p53 antibody (20 pg/ml) was also incubated for 10 hours. Blocking of nonspecific background staining was accomplished with normal sheep whole serum. Endogenous peroxidase was blocked with H202 and methanol. Biotinylated sheep antimouse IgG was incubated for 8 hours. Sections were incubated with streptavidin “bridge” and biotinylated horseradish peroxidase for 1 hour each. Peroxidase was detected with diaminobenzidine. Sections were gelatin mounted on slides for light microscopic evaluation. Control sections were prepared on each animal by performing immunohistochemistry, except that primary antibody was deleted. The remaining coronal slices from these rat brains were embedded in paraffin. Sections (6 pm) adjacent to those cut on the vibratome were stained with hematoxylin and eosin (H&E) for neuronal evaluation. RESULTS All serum arterial values of pH, pQ, and pC0, were within normal ranges.
In all
animals, the hemisphere contralateral to that of the MCA occlusion did not exhibit obvious cell damage, or hsp72 and p53 induction. In the ipsilateral hemisphere to the MCA occlusion,a homogeneous distribution of neuronal necrosis (- 80%) was found in the caudate putamen, lateral globus pallidus, and olfactory tubercle and adjacent structures. 1202
Luminal
and focal
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
neuronal necrosis (- 50%) was observed in piriform, on H&E staining (Fig. la,b).
Morphologically
RESEARCH
COMMUNICATIONS
insular, and parietal cortices, identified
intact hsp72 positive neurons were present in
the limbic and frontal cortices (Fig. 2a,b), and the temporal and occipital cortices of the peripheral zone, and absent from the necrotic core. primarily
Diffuse ~53 staining was evident and
localized to areas of neuronal necrosis in the ischemic center (Fig. 3a,b).
animals, more intense p53 staining was noted in the cortex than in the striatum.
In all
The cellular
source of the p53 staining was not apparent.
~53 was either, not detected (3 animals), or
weakly detected (2 animals) in morphologically
intact neurons, that were positively stained for
hsp72, in areas peripheral and adjacent to the ischemic center. ~53 was not evident in the contralateral hemisphere.
DISCUSSION Our data indicate that ~53 is expressed in regions of severe ischemic cell damage twelve hours after transient focal cerebral &hernia.
Areas encompassing necrotic neurons exhibit
positive staining for ~53, while areas, in the ipsilateral lesioned hemisphere, with intact neurons, fail to exhibit detectable levels of ~53, or in the region adjacent to the ischemic center may weakly stain for ~53.
Neither ~53 or hsp72 are expressed in the contralateral hemisphere.
Thus, the presence of ~53 is associated with neuronal necrosis. In light of our observations that ~53 is present coincident with necrotic neurons, we speculate, that the induction of ~53 after ischemia may facilitate cell death. Induction of ~53 in injured tissue, which promotes cell death, may be the converse of the loss of ~53 in tumor, which promotes uncontrolled proliferation (13). We have not yet determined, via DNA fragmentation studies (14), whether apoptosis is the mechanism of neuronal death.
Our data also cannot distinguish whether ~53 is simply
secondary to cell death, or causes cell death. However, our novel finding of ~53 in ischemic brain tissue associated with cell death, lends credence to a role for ~53 in promoting ischemic cell damage. hsp72 is present in cerebral tissue where ~53 is absent or reduced. ~53 binds to heat shock proteins of the 70kDa family (3,4).
Since hsp72 is present in morphologically
intact
neurons, and absent in necrotic neurons, this suggests that hsp72 may be active in protecting tissue, either by complexing
to ~53, and thereby regulating ~53 expression at the post
translational level, or by altering the morphology of ~53, thereby affecting ~53 function and antibody recognition. In vitro studies have shown that ~53 binds to the cognate (4), and to the inducible heat shock proteins of the 70kDa family (3). In the present study, both ~53 and hsp72 have been measured in vivo in non-tumored cerebral tissue. We employed a monoclonal antibody to
1203
182,No.3,1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIO
Fig. Coronalsections(Qm) stainedwith H&E were obtainedfrom all animals12 hours after 2 hoursof transientMCA occlusion,asdescribedin the text. Histologicaldamagewas presentin the right hemisphere (R) asa homogeneous distributionof neuronalnecrosisin the caudateputamen,lateralglobuspallidus,andolfactorytubercleandadjacentstructures.Luminal and focal neuronalnecrosiswas observedin the piriform, insularand parietalcokes. b. Enlargedareaof partialcortex, from thebox outlinedin Fig. la; neuronalnecrosiswasevident in approximately50% of the neurons. a x 8 , b x 103. 1204
INS
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fie. hsp72immunoreactivityin vibratomesections(50pm)was obtainedfrom the same animalasin Fig. 1. Morphologicallyintacthsp72positiveneuronswerepresentin limbic and frontal cortex andabsentfrom the necroticcorein the right hemisphere (R). b. Enlargedarea from the box outlinedin Fig 2a; showshsp72positiveneurons. a x 8 , b x 103. 1205
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIf
3NS
Fig. 3a. ~53 immunoreactivityin vibratomesections(5Orm) was obtainedfrom the same animalasin Fig. 1. Diffusep53 stainingwasevidentin the areasof neuronalnecrosislocated in the ischemiccenter. In this animal ~53 was weakly stained in areas containing morphologicallyintact neuronsand hsp72positiveneurons b. Enlargedarea from the box outlinedin Fig. 3a showsdiffuse~53 staining.The cellularsourceof the ~53 stainingwasnot apparent. a x 8 , b x103.
1206
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
hsp72. This antibody has no detectable cross reactivity with the cognate heat shock 70kDa protein, and does not stain in the contralateral non-ischemic hemisphere.
Thus, our data is
consistent with an association of ~53 with hsp72. In summary, we have found, for the first time, ~53 in ischemic cerebral tissue. The ~53 gene has been linked with inhibition
of cell proliferation as well as death in neoplastic cells.
Our data suggest, that induction of p53 may play a similar role in promoting
cell death in
ischemic tissue, as in tumor. REFERENCES
1.
Dippold WG, Jay G, DeLeo AB, and Khoury G Old LJ (1981) Proc. Natl. Acad. Sci. USA 78: 1695-1699.
2.
Benchimol S, and Pim D Crawford L (1982) EMBO J. 1:1055-1062.
3.
Pinhasi-Kimhi 185.
4.
Hinds PW, Finlay CA, Frey AB, and Levine AJ (1987) Mol Cell Biol.
5.
Nowak TS Jr (1985) J Neurochem.
6.
Sharp FR, Lowenstein DL, Simon R, and Hisanaga K (1991) J Cereb Blood Flow Metab. 11:621-627.
7.
Barbe MF, Tytell M, Gower DJ, and Welch WJ (1988) Science. 241-1817-1820.
8.
Chopp M, Chen H, Ho K-L, Dereski MO, Brown E, Hetzel FW, and Welch KMA (1989) Neurology. 39: 1396-1398.
9.
Rouach YE Resnitzlq D, Lotem J, Sachs L, Kimchi A, and Oren M (1991) Nature. 3521345-347.
10.
Zea Longa E, Weinstein PR, Carlson S, and Cummins R (1989) Stroke. 20:84-91.
11.
Vass K, Welch WJ, and Nowak TS JR (1988) Acta Neuropathol.
12.
Chopp M, Li Y, Dereski MO, Levine SR, Yoshida Y, and Garcia JH (1991) Acta Neuropathol. In Press.
13.
Finlay CA Hinds PW, and Levine AJ (1989) Cell. 57:1083-1093.
14.
Wyllie AH (1980) Nature.
0, Michalovitz,
Ben-Zeev, A, and Gren Moshe (1986) Nature. 320: 182p. 2863-2869.
45:1635-1641.
284:555-556.
1207
7:128-135.
