From the Research Service, Veterans Affairs Medical
Center, Denver, Colorado 80220, the § Department of
Medicine, University of Colorado Health Sciences Center,
Denver, Colorado 80220, and ¶ INSERM U145, Faculty of Medicine,
06107 Nice, France
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ABSTRACT |
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Rab proteins play a crucial role in the
trafficking of intracellular vesicles. Rab proteins are GTPases that
cycle between an inactive GDP-bound form and an active GTP-bound
conformation. A prerequisite to Rab activation by GTP loading is its
post-translational modification by the addition of geranylgeranyl
moieties to highly conserved C-terminal cysteine residues. We examined
the effect of insulin on the activity of geranylgeranyltransferase II
(GGTase II) in 3T3-L1 fibroblasts and adipocytes. In fibroblasts,
insulin increased the enzymatic activity of GGTase II 2.5-fold after
1 h of incubation, an effect that is blocked by perillyl alcohol, an inhibitor of prenyltransferases, but not by the
geranylgeranyltransferase I inhibitor, GGTI-298, or the
farnesyltransferase inhibitor, The family of Rab proteins is believed to play a crucial role in
the intracellular trafficking of vesicles along the endocytic and
exocytic pathways (1, 2). To date, over 30 members of this family have
been identified (3, 4). Although the exact role and mechanism of action
of Rab proteins remain to be elucidated, it appears that Rab proteins
are involved in the process of vesicular transport and precise
targeting of membranous vesicles to their docking and/or fusion sites
(5, 6). Rab proteins are GTPases that cycle between an inactive
GDP-bound form, cystosolic Rab, and an active GTP-bound conformation,
membrane-associated Rab, under the influence of Rab-associated guanine
nucleotide exchange factors and guanosine triphosphatase-activating
proteins (1-7). Although Rab protein activity is based on GTP loading,
a prerequisite to Rab activation is its post-translational modification
(4-6, 8, 9).
Post-translational modification of small molecular mass GTP-binding
proteins is accomplished by the isoprenylation of conserved cysteine
residues found on their C termini (10, 11). Ras and Rho proteins are
prenylated on a single cysteine residue of the CAAX box
(where C is cysteine, A is the aliphatic residue,
and X is methionine, serine, or glutamine for Ras
and leucine for Rho proteins) by farnesyltransferase
(FTase)1 and
geranylgeranyltransferase I (GGTase I), respectively. In contrast, Rab
proteins, which terminate with C-terminal cysteine motifs of CC or
CXC, are double prenylated by geranylgeranyltransferase II
(GGTase II) (10-13), an enzyme that catalyzes the attachment of a
geranylgeranyl moiety (four isoprenes, 20 carbons) to each C-terminal
cysteine residue. GGTase II, which is also known as Rab GGTase,
consists of a 60-kDa We have recently demonstrated that insulin promotes the phosphorylation
and activation of FTase, a ubiquitous enzyme responsible for the
attachment of farnesyl (3 isoprenes, 15 carbons) to
p21ras proteins (14, 15). Additionally, we found
that insulin, in a time-dependent fashion, stimulated the
phosphorylation of the FTase In the present study, we utilized Rab-3 and Rab-4, two substrates of
GGTase II, to evaluate the effect of insulin on GGTase II activity in
3T3-L1 fibroblasts and adipocytes. We observed that insulin promoted
the phosphorylation of the Materials--
Cell culture media and supplies were from Life
Technologies, Inc. and Gemini Biological Products (Calabasas, CA).
Radioisotopes were from NEN Life Science Products, and all standard
chemicals were from Sigma. Anti-Rab-3A monoclonal antibodies were from
Transduction Laboratories (Lexington, KY); polyclonal antibodies H-492
to Rab geranylgeranyltransferase II were a gift from Dr. Miguel Seabra (Imperial College School of Medicine, Norfolk, United Kingdom), and
protein G-PLUS/protein A-agarose was from Oncogene Research Products,
Inc. (Cambridge, MA). Rab-4 antibodies (produced against the protein)
were developed in the laboratory of Dr. Yannick Le Marchand-Brustel
(INSERM, Nice, France) (18), and the farnesyltransferase inhibitor,
Cell Culture--
3T3-L1 fibroblasts were grown to confluence in
fibroblast growth medium (Dulbecco's modified Eagle's medium
containing 5.5 mM glucose, 10% fetal calf serum, 50 µg/ml gentamicin, 0.5 mM glutamine, 0.5 µg/ml
Fungizone). Two days after confluence, fibroblasts were fed
differentiation medium (Dulbecco's modified Eagle's medium containing
25 mM glucose, 10% fetal calf serum, 50 µg/ml
gentamicin, 0.5 mM glutamine) plus differentiation mix (2.5 ml of 10× phosphate-buffered saline, 55 mg of
3-isobutyl-1-methylxanthine, 20 ml of deionized water, 250 µl of 49 mM dexamethasone, 2.5 mg of insulin). On day 4, adipocytes
were fed adipocyte growth medium (Dulbecco's modified Eagle's medium
with 25 mM glucose, 10% fetal calf serum, 50 µg/ml gentamicin, 0.5 mM glutamine) plus 100 nM
insulin. Cells were refed every 2 days with adipocyte growth medium and
used on days 10-12.
Measurements of Geranylgeranylated Rab Protein--
Confluent
cells were serum-starved for 24 h, incubated with or without
insulin (100 nM), and then lysed in 500 µl of buffer A
(150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 1 mM sodium vanadate, 1 mM
sodium phosphate, 1% Triton X-100, 0.05% SDS, 10 µg/ml aprotinin,
10 µg/ml leupeptin, 50 mM HEPES, pH 7.5). Crude lysates
were sonicated and centrifuged at 10,000 rpm. Total protein from the
resultant supernatant was determined by the bicinchoninic acid assay
(Pierce) and diluted to 1 mg/ml. Equal volumes of lysate and 4% Triton
X-114 were combined in a borosilicate glass tube, vortexed, and
incubated at 37 °C for 3 min. Solutions were kept at room
temperature until phases had separated. Equal volumes from each phase
were placed into separate 1.5-ml Eppendorf tubes, and Rab-3 or Rab-4
was immunoprecipitated using antibodies to Rab-3A or Rab-4. Relative
amounts of Rab proteins were determined by Western blotting followed by densitometry.
32P-Labeled Phosphorylation of the In Vitro Assay of GGTase II Activity--
GGTase II activity was
assayed in vitro using a modified method of Moores et
al. (19). In brief, cells were serum-starved for 24 h,
incubated for designated times with insulin (100 nM) in the
presence or absence of inhibitors, and lysed in 500 µl of buffer A. The in vitro enzymatic reaction was initiated by adding a
5-µl aliquot of diluted extract (0.5 mg/ml) to 45 µl of reaction
assay solution (5 mM MgCl2, 5 mM
dithiothreitol, 100 nM Rab-3 protein, 100 nM
tritiated geranylgeranyl pyrophosphate ([3H]GGPP) (15 mCi/mmol), 50 mM HEPES, pH 7.5) and incubated at 37 °C.
After 30 min the assay was stopped with 1 ml of ice-cold 1 M HCl in ethanol, and the samples were placed on ice for 15 min. Solutions were filtered through Whatman GF/C glass-fiber filters. Each filter was air-dried, placed in a scintillation vial with 3 ml of
scintillation fluid, and quantified by liquid scintillation spectrometry. The in vitro GGTase II assay was linear with
respect to time and extract protein.
In Vivo Assay of Labeled Rab-3 Using [3H]Mevalonic
Acid--
Confluent 3T3-L1 fibroblasts were placed in serum-free
medium containing lovastatin (2 µg/ml) and
[3H]mevalonolactone (10 µCi) for 24 h. Cells were
then incubated for 1 h with or without insulin (100 nM), lysed, and normalized for protein. Rab-3 proteins were
immunoprecipitated from the aqueous and detergent fractions and
analyzed by SDS-PAGE. Proteins and labeled product were determined by
Western blotting and autoradiography, respectively, and quantified by densitometry.
Triton X-114 Extraction of in Vitro Labeled Rab-3 Using
[3H]GGPP--
Confluent cells were serum-starved for
24 h and then incubated with or without insulin (100 nM) for 1 h, lysed, and normalized for protein. The
in vitro reaction was initiated by adding a 5-µl aliquot
of diluted extract (0.5 mg/ml) to 45 µl of reaction assay solution (5 mM MgCl2, 5 mM dithiothreitol, 100 nM Rab-3 protein, 100 nM tritiated
geranylgeranyl pyrophosphate ([3H]GGPP) (15 mCi/mmol), 50 mM HEPES, pH 7.5) and incubated at 37 °C. After 30 min
the assay was stopped with 50 µl of ice-cold 1 M HCl in
ethanol, and the samples were placed on ice for 15 min. Samples were
diluted to 800 µl with 1× phosphate-buffered saline and incubated
with an equal volume of 4% Triton X-114 for 3 min. Rab-3 proteins were
then immunoprecipitated from the aqueous and detergent fractions and
analyzed by SDS-PAGE. Proteins and labeled product were determined by
Western blotting and autoradiography, respectively.
Statistical Analysis--
Data were analyzed by Student's
paired or unpaired t test with a p value of
<0.05 considered significant.
Insulin, a potent modulator of FTase activity in various tissues,
increases the amounts of cellular farnesylated
p21ras (14-16, 20). To determine whether insulin
can also stimulate other prenyltransferases, we examined its effect on
GGTase II activity. Initially, we performed an in vitro
assay of GGTase II activity in control and insulin-treated 3T3-L1
fibroblasts. The cells were exposed to insulin for up to 2 h, and
their lysates were then used as a source of enzyme (GGTase II) as
described under "Experimental Procedures." Enzymatic activity of
GGTase II is expressed as the amount of [3H]GGPP
(counts/min) attached to the recombinant Rab-3 in vitro. Insulin significantly increased the enzymatic activity of GGTase II
1.5-fold (p < 0.05) after 45 min of incubation and
2.5-fold (p < 0.01) after 1 h of incubation (Fig.
1). This effect of insulin was blocked by
1 mM POH, an inhibitor of prenyltransferases, but not by
GGTI-298, an inhibitor of GGTase I, or -hydroxyfarnesylphosphonic acid. Concomitantly, insulin stimulated the phosphorylation of the GGTase II
-subunit without any effect on the GGTase II
-subunit. At the same time, insulin also increased the amounts of
geranylgeranylated Rab-3 in 3T3-L1 fibroblasts from 44 ± 1.2% in
control cells to 63 ± 3.8 and 64 ± 6.1% after 1 and
24 h of incubation, respectively. In adipocytes, insulin increased
the amounts of geranylgeranylated Rab-4 from 38 ± 0.6% in
control cells to 56 ± 1.7 and 60 ± 2.6% after 1 and
24 h of incubation, respectively. In both fibroblasts and
adipocytes, the presence of perillyl alcohol blocked the ability of
insulin to increase geranylgeranylation of Rab-4, whereas GGTI-298 and
-hydroxyfarnesylphosphonic acid were without effect, indicating that
insulin activates GGTase II. In summary, insulin promotes phosphorylation and activation of GGTase II in both 3T3 L1 fibroblasts and adipocytes and increases the amounts of geranylgeranylated Rab-3
and Rab-4 proteins.
INTRODUCTION
Top
Abstract
Introduction
References
-subunit and a 38-kDa
-subunit heterodimer
that are closely associated with a 95-kDa protein called the Rab escort
protein (10-13). The Rab escort protein binds together with the
/
dimer of GGTase II as a holoenzyme complex and functions to
present Rab proteins to GGTase II for prenylation.
-subunit (16), an event that correlated
well with insulin-stimulated FTase activity (16). Because FTase and
GGTase I share the same
-subunit (17), one could assume that the
enzymatic activity of GGTase I might also be regulated by insulin.
However, the potential influence of insulin on GGTase II, which has
approximately 20-30% homology with FTase and GGTase I, has not
been verified experimentally.
-subunit of GGTase II and an increase in
GGTase II activity. Consequently, we found insulin increased amounts of
geranylgeranylated Rab-3 and Rab-4 in the insulin-challenged cells.
EXPERIMENTAL PROCUDURES
-hydroxyfarnesylphosphonic acid (
-HFPA), was from Biomol
(Plymouth Meeting, PA). (S)-(
)-perillyl alcohol (POH) was
from Aldrich; the GGTase I inhibitor GGTI-298 was a gift from Dr.
Saïd Sebti (University of South Florida), and lovastatin was
from Merck. All supplies and reagents for SDS-PAGE were from Bio-Rad,
and the enhanced chemiluminescence kit (ECL) was from Amersham
Pharmacia Biotech.
- and
-Subunits of GGTase II--
Cultured cells were serum- and
phosphate-starved for 6 h and then incubated at 37 °C
overnight with 250 µCi of [32P]orthophosphate (10 mCi/mmol). Cells were then incubated for various times with or without
insulin (100 nM). Lysates (in buffer A) were sonicated and
centrifuged. Protein concentrations were diluted to 1 mg/ml. GGTase II
- and
-subunits were immunoprecipitated with GGTase
- or
-subunit rabbit antiserum, analyzed by SDS-PAGE, and
visualized by autoradiography.
RESULTS
-HFPA, an inhibitor of
FTase.
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Fig. 1.
Effect of insulin and POH on the activity of
GGTase II. Confluent 3T3-L1 cells were serum-starved for 24 h, incubated with insulin (100 nM) in the absence
(solid line) or presence (dotted line) of POH (1 mM) for indicated times, lysed, and normalized for protein.
A 5-µl sample was incubated at 37 °C with 45 µl of buffer
containing [3H]GGPP and Rab-3 protein for 30 min as
described under "Experimental Procedures." The reaction was stopped
and the solution was filtered through Whatman GF/C glass-fiber filters.
Filters were placed in 3 ml of scintillation fluid, and labeled Rab-3
was quantified by spectrometry. Results represent mean ± S.E. of
three independent experiments performed in duplicate. *,
p < 0.05; **, p < 0.01.
Because insulin-stimulated activation of FTase has been shown to be
related to the phosphorylation of its -subunit (16), we examined
whether or not insulin could also promote the phosphorylation of GGTase
II. Insulin appears to induce a rapid phosphorylation of the GGTase II
-subunit, without any effect on its
-subunit (Fig.
2). Insulin-stimulated phosphorylation of
the
-subunit was detectable by 10 min and clearly evident by 1 h. Taken together, these data indicated that, as with FTase,
insulin-stimulated activation of GGTase II correlated with its
phosphorylation.
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Because we previously showed that the activation of FTase by insulin resulted in significant increases in the amount of farnesylated p21ras (14-16), we asked if insulin-stimulated augmentation in GGTase II activity might result in increases in the amount of geranylgeranylated Rab proteins. To address this possibility, we examined the effect of insulin on the geranylgeranylation of Rab-3 in 3T3-L1 fibroblasts and PC-12 cells, using the method of Triton X-114 extraction (21).
To validate this technique, we needed to establish that only prenylated Rab-3 protein was extracted into the detergent phase. To this end, we extracted the lysis buffer A containing unprocessed (bacterially expressed) Rab-3 protein with Triton X-114 and determined that unprocessed (nonprenylated) Rab-3 was detected only in the aqueous phase (Fig. 3A). Conversely, endogenous geranylgeranylated Rab-3 was detected only in the detergent phase (Fig. 3, B and D). In these experiments, the cells were incubated with lovastatin (2 µg/ml) to inhibit isoprenoid synthesis and then with 10 µCi of [3H]mevalonate, a precursor of geranylgeranyl moiety that enters the synthetic pathway downstream of the lovastatin block. The cells were then lysed and extracted with Triton X-114. Newly labeled geranylgeranylated Rab-3 was detected only in the detergent phase (Fig. 3D), whereas Rab-3 detected in the aqueous phase by Western blotting (Fig. 3B) was not prenylated. Finally, we determined that the recombinant Rab-3 that was prenylated in vitro with labeled geranylgeranyl pyrophosphate was recovered only in the detergent phase (Fig. 3, C and E).
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Subsequently, using the Triton X-114 extraction method we found that
geranylgeranylation of Rab-3 was significantly enhanced by insulin in
both 3T3-L1 fibroblasts (Fig. 4) and
PC-12 cells (not shown). Insulin significantly increased the amounts of
geranylgeranylated Rab-3 in 3T3-L1 fibroblasts from 44 ± 1.2% in
control cells to 63 ± 3.8% (p < 0.01) after
1 h of incubation and to 64 ± 6.1% (p < 0.01) after 24 h of incubation. POH but not GGTI-298 or -HFPA blocked the effects of insulin on geranylgeranylation of Rab-3. Taken
together, these results suggest that geranylgeranylation of Rab-3 is
mediated by the activity of GGTase II and augmented by insulin action.
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Because Rab-4 has been implicated in the mechanism of the
insulin-stimulated glucose uptake (22, 23), we were also interested in
the effect of insulin on Rab-4 prenylation. In the next series of
experiments we examined the effect of insulin on the
geranylgeranylation of Rab-4 in 3T3-L1 adipocytes. Insulin
significantly increased the amounts of geranylgeranylated Rab-4 from
38 ± 0.6% in control cells to 56 ± 1.7%
(p < 0.01) after 1 h of incubation and to 60 ± 2.6% (p < 0.01) after 24 h of incubation
(Fig. 5). Similarly to the experiments
with Rab-3, the presence of POH (1 mM) blocked the ability
of insulin to increase geranylgeranylation of Rab-4, whereas GGTI-298
and -HFPA were without effect. Interestingly, the ability of insulin
to increase the amounts of geranylgeranylated Rab-4 was also inhibited
by a mitogen-activated protein kinase kinase inhibitor, PD 98056, but
not by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (Fig.
6).
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DISCUSSION |
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The present experiments demonstrate that insulin-stimulated
phosphorylation and activation of GGTase II are accompanied by increases in the amounts of geranylgeranylated Rab proteins in fibroblasts, adipocytes, and PC-12 cells. This aspect of insulin action
appears to be similar to its ability to phosphorylate and activate
FTase and to increase the amounts of farnesylated p21ras
(14-16). We have previously demonstrated that insulin promotes the
phosphorylation of the -subunit of the FTase in association with
augmented FTase activity (16). Because the same
-subunit belongs to
GGTase I as well (17), one could predict that insulin would also
activate GGTase I. Our ongoing studies support this prediction.2 Thus, based on
the current findings (activation of GGTase II) and previous
observations (activation of FTase), we conclude that insulin is a major
regulator of isoprenylation of the small molecular mass GTP-binding
proteins. To further strengthen this conclusion, the following
questions must be addressed. What is the precise mechanism of the
effect of insulin on prenyltransferases? What is the physiological
significance of this aspect of insulin action?
Our previous work has addressed certain elements of both questions. We
have determined that the effect of insulin on FTase is specific. Other
growth factors such as insulin-like growth factor 1, epidermal growth
factor, and platelet-derived growth factor failed to promote FTase
phosphorylation and stimulate its activity (15). Furthermore, insulin
appears to promote the phosphorylation of the FTase -subunit in a
positive feedback fashion (16). We found that the ability of insulin to
activate the Ras-mitogen-activated protein kinase pathway was critical
for the subsequent phosphorylation and activation of FTase. The latter,
in turn, increased the amounts of farnesylated p21ras proteins
making them available for activation by GTP loading (15, 20). It
appears that the effect of insulin on GGTase II also involves
phosphorylation of the
-subunit of this enzyme (Fig. 2). Moreover,
the ability of insulin to increase the amounts of geranylgeranylated
Rab-4 was blocked by the inhibitor of the mitogen-activated protein
kinase kinase pathway (Fig. 6), emphasizing a similarity to the
mechanism of the effect of insulin on FTase and farnesylation of Ras
(16).
The physiological and/or pathophysiological role of the insulin-induced modulation of isoprenylation of small molecular mass GTPases constitutes the second major question in this field. At present, we can only state that insulin, via its ability to regulate the amounts of prenylated Ras and Rab, creates a certain background that is responsible for the magnitude of activation of these GTPases by their appropriate stimuli. Interestingly, cells that are derived from insulin receptor-deficient mice exhibited normal basal levels of farnesylated p21ras, although they failed to respond to an insulin challenge (15). Moreover, even using potent inhibitors of FTase (15, 20) and GGTase I and II (present study) we were unable to deplete the cells of the prenylated Ras or Rab within a time frame of our experiments (24 h). These observations suggest that insulin is only responsible for augmenting and modulating prenyltransferase activity over the basal rates of their activity. Clearly, additional experiments are needed to gain more insight into the mechanism of the insulin action on FTase and GGTase I and II.
Geranylgeranylation of Rab proteins is believed to be the first step in their post-translational modification, allowing them to interact with cellular membranes (8-11). Reversible phosphorylation-dephosphorylation of Rab-4 and Rab-1 appears to add an extra layer of control that determines cellular localization of these proteins during the cell cycle (interphase versus mitosis) (24, 25). Prenylated Rab-4 and Rab-3 have been found in both soluble and membranous cellular fractions (26), suggesting that prenylation itself is not solely responsible for the membrane association. Moreover, van der Sluijs et al. (25) have demonstrated that 82% of the newly geranylgeranylated wild-type Rab-4 was found in the cytosol of the mitotic Chinese hamster ovary cells. Mutation of Ser-196 to glutamine or aspartic acid completely prevented Rab-4 phosphorylation in mitotic cells and blocked its appearance in the cytosol (25). Association of Rab proteins with the GDP-dissociation inhibitor protein represents an additional mechanism that regulates the interaction of Rab proteins with cellular membranes (26-29). Chinni et al. (30) have demonstrated that even though the release of GDP-dissociation inhibitor from the intracellular membranes did not affect the cellular distribution of Rab-4 (only Rab-5 was affected) it was associated with the insulin-stimulated glucose transporter (GLUT)-4 membrane trafficking.
Whether or not geranylgeranylation plays a significant physiological role in the mechanism of Rab-4 involvement in the insulin-stimulated glucose uptake remains unknown. Our previous experiments with inhibition of prenylation by lovastatin (31) suggest that insulin essentially stimulates glucose transport normally in cells pretreated with lovastatin for up to 24 h. However, within the time frame of the experiments (24-48 h) inhibitors do not deplete the pool of prenylated Rab-4 below the basal levels that may be sufficient to assure the normal function of Rab-4 in response to insulin. Longer term experiments in the future, both with insulin and the GGTase II inhibitors, are needed to examine this relationship.
Although the physiological and pathophysiological significance of the
ability of insulin to increase the amounts of geranylgeranylated Rab-3
and Rab-4 remains unknown, new data presented here indicate that
insulin is an important regulator of the cellular pool of prenylated
Rab proteins available for GTP loading and carrying out their
specific biological functions.
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FOOTNOTES |
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* This work was supported by the Veterans Affairs Medical Center Research Service, the Foundation for Biomedical Education and Research, the American Heart Association (fellowship to M. L. G.), and the Institut National de la Recherche Médicale APEX 97-01 (to Y. L. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Research Service
(151), Veterans Affairs Medical Center, 1055 Clermont St., Denver, CO
80220. Tel.: 303-393-4619; Fax: 303-377-5686; E-mail:
DRAZNINB{at}den-res.org.
The abbreviations used are:
FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; -HFPA,
-hydroxyfarnesylphosphonic acid; POH, (S)-(
)-perillyl alcohol; GGPP, geranylgeranyl
pyrophosphate; PAGE, polyacrylamide gel electrophoresis.
2 M. L. Goalstone, M. R. Stjernholm, J. W. Leitner, and B. Draznin, unpublished observations.
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REFERENCES |
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