CHARACTERIZATION OF HYPOTHALAMIC SUBCELLULAR PARTICLES CONTAINING LUTEINIZING HORMONE RELEASING HORMONE AND THYRUTROPIN RELEASING HORMONE AYALLABARNEA,NIRABEN-JONATHAN and JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Physiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, TX 75235, U S A . (Received 23 December 1915. Accepted 24 February 1976)
Abstract-The 900 g supernatant fluid prepared from male rat hypothalamic homogenates was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone and luteinizing hormone releasing hormone in the gradient fractions were quantified by radioimmunoassays. TRH was associated with two populations of particles separable by means of nonequilibrium density centrifugation (l00,ooO g for 30 min). However, after 'equilibrium' centrifugation (100,ooO x g for 180 min), a single peak of TRH was observed at 1.07M-sucrose. Hypo-osmotic shock as well as treatment with 0.1% Triton X-100or O.lYa deoxycholate (DOC) released TRH from both sets of particles. LRH, as TRH, was associated with two populations of particles which were separable by means of nonequilibrium density gradient centrifugation. After 'equilibrium' centrifugation, both sets of LRHcontaining particles banded at 1.27 M-sucrose as a single symmetrical peak. Although 0.1", Triton X-100 released LRH from both populations of particles, hypo-osmotic shock or 0.17; DOC released LRH only from the large LRH-containing particles. The small LRH-containing particles were resistant to hypo-osmotic shock and to 0.1:; DOC. Based on these criteria, it is concluded that in hypothalamic homogenates the TRH-containing particles and the large LRH-containing particles are synaptosomes. The small LRH-containing particles may be of different cellular and/or subcellular origin.
TRH AND LRH in hypothalamic homogenates are et a/., 1974; ZIMMERMAN et a/., 1974; BAKERr t a/., associated with subcellular particles displaying sedi1975; S~TALO~t of.. 1975). However, it seems that mentation characteristics similar to those of particles these criteria are insufficient for a conclusive identifiet a/., 1975). In brain containing DA and NE (BARNEA cation of the T R H - and LRH-containing particles as homogenates, DA and NE are confined in particles synaptosomes. known as synaptosomes, which are pinched-off preThe standard preparations of synaptosomal fracsynaptic axonal terminals (DE ROBERTIS& DE LORES tions (DEROBERTIS& DELORES ARNAIS,1969; WHITARNAIS,1969; WHITTAKER, 1969). Therefore, it could TAKER, 1969) are not homogenous with regard to their be assumed that the particles containing T R H and content of other subcellular organelles. These fracLRH are also synaptosomes. This assumption is tions are contaminated with broken axonal segments further substantiated by the observations that in addi- (LEMKEY-JOHNSON & DEKIRMENJIAN. 1970) as Well as tion to the regulation of pituitary function, T R H with membranes of the endoplasmic reticulum and (DYER& DYBALL,1974; KELLERer a/., 1974; MET- fragments of the Golgi complex (FLEISCHERei id.. CALF, 1974: RENAUD er a/., 1975; WEI & SIGEL,1975) 1969; GURDet al., 1974). Moreover, it has been shown as well a s LRH (Moss & MCCANN, 1973; PFAFF, (COTMAN er al., 1971) that portions of the plasma 1973; DYER& DYBALL,1974; RENAUDet a/.. 1975) membranes from glial cells sediment in the crude have direct effects on the brain. Thus, these peptides mitochondria1 fraction and have an isopycnic banding resemble neurotransmitters in their mode of action. density on sucrose gradients very similar to that of Furthermore, T R H is widely distributed throughout synaptic plasma membranes and intact synaptosomes. the brain (BROWNSTEIN ef a/., 1974; OLIVER ef a/.. Therefore. in order to identify the TRH- and LRH1974; WINTERSef a/., 1974). and a neuronal localiza- containing particles, one cannot rely solely on their tion of LRH in the hypothalamus has been demon- sedimentation characteristics. A well-documented strated by immunohistochemical techniques (BARRY property of synaptosomes is their susceptibility to hypo-osmotic shock (DE ROBERTIS & DE LORES ARNAIS, 1969; WHITTAKER, 1969). We have previously Ahbreriurions used: DA, dopamine; DOC. sodium deoxycholate; LRH. luteinzing hormone releasing hor- reported that hypo-osmotic conditions affect particumone; PBS, 0.01 M-sodium phosphate buffer containing late TRH (BARNEAer (11.. 1975) in a manner similar 0.14 M-NaCI and O.Olo< merthiolate, pH 7.0; TRH. thyro- to that reported for synaptosomes containing catecholamines (DEROBERTIS& DE LORESARNAIS 1969). tropin releasing hormone. 477
478
AYALLA BARNEA, NIRABEN-JONATHANand JOHN C PORTER
whereas a significant portion of the particle-bound LRH was found to be resistant t o hypo-osmotic treatment (BARMA er d..1975). Moreover, some of the particles containing LRH and T R H differed in their sedimentation characteristics. In this investigation. an extensive study was made of the kinetics of the distribution of hypothalamic LRFI- and TRH-containing particles o n sucrose density gradients. In addition, the effects of hypo-osmotic shock and various detergents o n the different populations of these particles were tested.
standards. producing a final detergent concentration of OS",,. The recobery of TRH and LRH from the gradients was 88 2.9"" and 93 k 4.3",, (mean k S.E.M. of 1 1 gradients). respectively. DA and NE were quantified by means of a radioenzymatic assay (BTN-JOKATHAN & PORTER.i n press).
Sources of materials. Na,-EDTA (Baker, Phillipsburg. NJ), LRH and TKH (Beckman Instruments. Inc.. Palo Alto. CA). DOC (Nutritional Biochemical Corp.. Cleveland, OH). sucrose (Schwarz,'Mann. 909530. Orangeburg. NY), Triton X-100 (Sigma. St. Louis, MO), filters of mixed esters of cellulose (Millipore Corp.. Bedford, MA). The antiserum to TRH was prepared in our laboratory as described previously (ESKAYer ul.. 1976). The antiserum to LRH was a gift from Dr. G. "VENDER.
MATERIALS AND METHODS Adult male rats of the Long-Evans strain (200-250g) were used. The animals were decapitated. and their brains were removed rapidly and placed in ice-cold 0.15 M-NaCI. All operations were performed at W"C, and all solutions containing sucrose also contained 10pmol of CaCI, per liter. Three hypothalami were homogenized in 0.75 ml of 0.32 M-sucrose-10 pM-CaCI, with a Dual1 tissue grinder and a Teflon pestle (Kontes. K-885480,Vineland, NJ) using ten up-and-down strokes. Homogenates were diluted with I vol of homogenizarion medium and centrifuged at 900 g for 10min. One ml of the 9009 supernatant fluid was layered on a continuous sucrose density gradient prepared as described previously (BARNEAet al., 1975). The tubes were centrifuged at 29,000 rev./min (100,ooO x gaV) for designated times at 4'C in a Beckman Model L2-65 Ultracentrifuge using an SW 40 rotor (Beckman Instruments, Inc., Palo Alto, CA). A t the end of the run, 0.3 ml fractions were collected using an ISCO Model 640 Density Gradient Fractionator (Instrument Specialties Co., Lincoln, NE). In order to facilitate comparison of the locations of the various substances, it was assumed that the concentration of sucrose changed linearly on the gradient. Retention of particles on celltilose ester filters. Particlebound peptides were trapped on cellulose ester filters essentially as described for synaptosomes containing NE (BALDESSARINI& VOGT, 1971). Filters were pre-soaked in I", EW-PBS. pH 7.0. for at least 2 h. A combination of two filters was used. A filter of pore size 0.8 pm was placed on top of a filter having 0.22 pm pore size, and both were placed on a filter holder (Hoeffer Scientific Instruments. San Francisco, CA). The apparatus was attached to a vacuum line, and the excess solution aspirated. The 9009 supernatant fluid was diluted with 1 vol of 0.32 M-SUCTOSe. and to this mixture. 0.1 vol of a 1OX Hank's salt solution (PAUL.1974) was added. Samples of 0.5 ml were filtered and then washed with 2ml of 0.32~-sucrose.Particles retained on the filters were extracted by sonication for 15 min in an ice bath using a Megason Sonicator (Megason Ultrasonic Instruments. Farmingdale, NY). Assays. Samples were analyzed by radioimmunoassay for TRH (ESKAYet a/., 1976) and LRH (NETTet al., 1973) using synthetic peptides as reference standards. In order to solubilize the particle-bound TRH and LRH. Triton X-100 was added to all samples as well as to the reference
'The boundaries of the peaks were measured at half their heights and are indicated in brackets.
RESULTS
Kinetics o f t h e disrribirrioii of T R H - and LRH-coritaining particles on density gradients As noted previously (BARNEAer ul., 1975). T R H and L R H are associated with particles which can be partially separated by differential and density gradient centrifugation. The following experiments were designed t o answer the question whether these particles differ in size and/or density. The 900g supernatant fluid was fractionated o n gradients ranging from 0.4 to 1.4M-sucrose for 30, 90, or 180 min. A progressive migration along the gradients of the particles containing T R H a n d LRH as a function of centrifugation time was noted. After 30 min, T R H distributed as two distinct peaks located at 0 . 6 (~0 . 5 0 . 7 0 ~ ) 'a n d a t 1.07 M (1.W1.15 M) sucrose (Fig. 1). After 90min, T R H distributed as a single asymmetrical peak a t I .04M-sucrose; whereas after 180 min, a symmetrical peak was noted at 1.07 M (0.99-1.15 M). LRH was distributed along the gradients after 30min of centrifugation as two peaks located at 0.85 M (0.72-0.97 M) and 1.18 M ( 1.1 @ 1.27 M) sucrose (Fig. 1). After 90min, the 0.85 M peak disappeared, and LRH banded as a single peak at 1.20 M (1.06-1.34M). After 180 min, the peak was located at 1.27 M (1.18-1.37 M) sucrose. The kinetics of the migration of the TRH- and LRH-containing particles was further evaluated using sucrose gradients ranging from 0.C1.6 and 0 . 9 - 1 . 6 ~ (Figs. 2 and 3). Regardless of the density of the sucrose used, single peaks of T R H and of LRH were noted at 1.07 (0.97-1.20~) and 1 . 2 7 ~ (1.17-1.37 M), respectively, after 180min of centrifugation. Further centrifugation of the 0.61.6 M gradients for 300min did not alter the positions of T R H and L R H o n the gradient, indicating that conditions of 'equilibrium' centrilugation were approached by 180 min (DEDUVE,1971). These results also show that T R H and LRH are compartmentalized in separate subcellular particles. The TRH-containing particles are less dense than those containing LRH. Although all particles containing a given peptide are of similar density, they are heterogenous in size.
Hypothalamic particles containing LRH and TRH
480
1
Hypo-osmotic shock (Fig. 4) and treatment with DOC (Fig. 5) resulted in the disappearance of the symmetrical peaks of DA and NE. Furthermore, hypo-osmotic shock and to a larger extent DOC treatment reduced the magnitude of the DA shoulder, whereas only DOC was effective in reducing that of NE. The DA and NE released by these treatments were recovered at the top of the gradients. These results indicate that the shoulder is comprised of particle-bound DA and NE. (The gradients containing Triton X-100 were not analyzed for DA and NE since this detergent interfered with the assay.) Hypo-osmotic shock (Fig. 6) and Triton X-100 (Fig. 7) caused complete disappearance of the 0 . 6 ~ and 1.07 M peaks of TRH and the appearance of TRH at the top of the gradients. However, we consistently noted a small peak of TRH at about 0.5~-sucrose following hypo-osmotic shock. DOC treatment (Fig. 8) was not quite as effective as Triton X-100 (Fig. 7) in releasing particle-bound TRH. Homogenization with Triton X-100 also resulted in the disappearance of the two peaks of LRH (Fig. 7). Hypo-osmotic shock abolished the 1.18 M peak of LRH, whereas the magnitude of the 0.85 M peak was either unaltered (Fig. 6) or somewhat reduced in some experiments. D O C treatment resulted in reduction of the 1 . 1 8 ~peak of LRH and had no apparent effect
TRH
-,-
419
~
0.32 0.4
SUCROSE CONCENTRATION ( M )
FIG.1. Kinetics of the distribution of TRH- and LRH-containing particles on sucrose gradients. The 900 g supernatant fluid, prepared from hypothalami homogenized in 0.32 M-sucrose-10 pM-CaCl,, was layered on sucrose gradients ranging from 0.4 to 1.4 M. The gradients were centrifuged at 100,000g for 30, 90, or 180min.
Differential eflects of hypo-osmotic shock and detergents on parricle-bound 7RH and lRH and synaptosomcs containing D A and N E
Hypothalami were homogenized in (1) hypo-osmotic solution (10pM-CaCI,), (2) iso-osmotic solution (0.32 M-SUCrOS-510pM CaCI,), (3) 0.1"6 DOC in 0.32 M-sucrose-10 pM-CaCI,, or (4)0.10; Triton X-100 in 0.32 M-sucrose-10 pM-CaCI,. The 900 g supernatant fluids were fractionated on gradients ranging from 0.4-1.4 M-sucrose for 30 min, and the profiles of TRH, LRH, DA, and NE were determined. After fractionation of the 9009 supernatant fluid prepared in 0.32 M-sucres, DA as well as NE distributed as two peaks along the gradient (Fig. 4).One asymmetrical peak with a broad shoulder was present at the top of the gradient. This shoulder was located in gradient fractions corresponding to the 0 . 6 ~peak of TRH (Fig. 1). The second peak, which was symmetrical. was located in the same fractions as the 1.07 M peak of TRH. DA and NE distributed similarly to each other (Fig. 4). \ I
'-:
I
GRADIENT FRACTION I
1-1
0'32 o-6
SUCROSE CONCENTRATION (MI
1.6
FIG.2. Kinetics of the distribution of TRH- and LRH-containing particles o n sucrose gradients ranging from 0.6 to 1.6 %I. See Fig. I for details.
A ~ A L LBARYFA. A NIRABEN-JONATHA~ and JOHN C . PORTER
4x0
o n the 0.85 M peak (Fig. 8). Unlike free T R H , DA, or NE. free LRH was not demonstrable on the graO--a
dients. The disparity in the degree of solubilization of particle-bound T R H and LRH is further demonstrated in the following experiment. The 9009 supernatant fluid was incubated with increasing concentration of detergent for 10 min at 4 C, and the particle-bound peptides were trapped on cellulose ester tilters. As shown in Table I, T R H was partially solubilized in 0.1", DOC and was completely solubilized in 0.5",
30 H I N
n9 0 M I N b - 4 180 Y I N
TABLE 1. DIFFERENTIAL EFFECTS PARTICLE-BOUND LRH
OF AND
Concentration of detergent
GRADIENT FRACTION
c-l-
''.O
4 SUCROSE CONCENTRATION (M)
1.6
FIG.3. Kinetics of the distribution of TRH- and LRH-containing particles on sucrose gradients ranging from 0.9 to 1 . 6 ~ See . Fig. 1 for details.
DETERGENTS
Bound Bound LRH TRH
Treatment
P")
r")
C'")
None Triton X-100 Triton X-100 DOC DOC DOC
0 0.02
loo* 58 1 96 90
100 69 2 94 50 2
0.10 0.02
0.10 0.50
34
The 9009 supernatant fluid. diluted as described in Materials and Methods, was incubated with increasing concentrations of Triton X-100 or DOC for 10 min at 4 C . Particle-bound LRH and TRH were trapped on cellulose ester filters and then extracted with 2 ml of 90":, methanol. The methanol was evaoorated to dryness. the residual peptides redissolved in 2 ml PBS. and samples of 150 ,ul were assayed for TRH or LRH as described. Results are the mean of duplicate experiments. * Percent'of controls: bound LRH In conti-ols = I5 pg; bound TRH = 8Opg.
GRADIENT FRACTION t : 0.32 0.4
I
ON
TRH
U
1.4 0.32 0 4 SUCROSE CONCENTRATION
I 1.4
(MI
FIG.4. ERect of hypo-osmotic shock on the gradient profiles of DA and NE. The 900 g supernatant fluid, prepared from hypothalami homogenized in 0.32 M sucrose-10 ~ M - C A C(iso-osmotic) I~ or in 10 IIMCaCI, (hypo-osmotic). was fractionated on gradients ranging from 0.4 to I .4 M-sucrose at 100.000 y for 30 min.
Hypothalamic particles containing LRH and T R H
-"
.-c0
48 1
1.2-
e
t L
0 D
p
0.8 -;
0.4
-! L
O/0
20 1 ;
0.32 0.4
40
0
20
G R A D I E N T FRACTION i +I 1.4 0.32 0.4 SUCROSE CONCENTRATION ( M I
40 I 1.4
FIG.5. Effects of DOC on the gradient profiles of DA and NE. The 900 g supernatant fluid, prepared from hypothalami homogenized in 0.32 M-sucrose-10 pt-CaCI, or in 0.32 M-sucrose-I0 pM-CaC1, containing 0.1% DOC, was fractionated on gradients ranging from 0.4 to 1.4 M-sucrose at 1 0 0 , g~ for 30 min. DISCUSSION DOC. However, significant solubilization of particlebound LRH was effected only in 0.5"/, DOC. In conThe TRH-containing particles observed previously trast-to DOC, Triton X-100 was equally effective in (BARNEAet al., 1975; WINOKUR & UTIGER,1975) as solubilizing TRH and LRH. Partial solubilization of ,well as In this study exhibit properties resembling both peptides occurred in 0.02:4 Triton and complete synaptosomes. For example. hypo-osmotic shock results in the release of most of the TRH contained solubilization occurred in 0.1%.
/-I 0.32 0.4
1H
I
1.4 0.320.4
1.4
SUCROSE CONCENTRATION ( M I
FIG.6. Effect of hypo-osmotic shock on the gradient profiles of L R H and T R H . See Fig. 4 for details
182
AVALLA BARSEA.NIRABEN-JOKATHAN and JOHN C. PORTER
FIG. 7. Effects of Triton X-100 on the gradient profiles of LRH and TRH. The 9009 supernatant fluid, prepared from hypothalami homogenized in 0.32 M-SuCrOSe-10 pM-CaCI, or in 0.32 M-Sucrose-10 pM-Caa, containing 0.1% Triton X-100,was fractionated on gradients ranging from 0.4 to 1.4 M-SUCTOSe at 100,ooO g for 30 min.
in the two populations of particles as well as a large fraction of the synaptosomal DA and NE. This characteristic is in accord with that reported for synaptosomes containing acetylcholine and catecholamines (DE ROBERTIS & DE LORESARNAIS,1969). Furthermore, Triton X-100, which solubilizes the presynaptic portion of acetylcholine-containing synaptosomes (FISZER & DE ROBERTIS,1967), solubilized particle-bound TRH ; and DOC dissolved particle-bound TRH as well as synaptosomal DA and NE. Hence, these physical properties strongly support the conclusion that the particles containing TRH are synaptosomes. In the present study, LRH, as TRH, was found in two populations of particles separable by means of nonequilibrium density gradient centrifugation. Moreover, these particles banded as a single peak when subjected to ‘equilibrium’ density centrifugation indicating that they differ in size but are similar in density. The observation that after ‘equilibrium’ density centrifugation the LRH- and TRH-containing particles banded at distinguishable densities (LRH at 1.27 M and TRH at 1.07 M) does not negate the possibility that LRH is also localized in synaptosomes. Such a possibility is in accord with the findings that synaptosomes containing GABA, histamine, and NE vary in sedimentation characteristics (KUHARet al., 1971). Nonetheless, other physical properties of the small LRHcontaining particles differed markedly from those of synaptosomes containing TRH and catecholamines. Although LRH associated with the
larger particles was released by hypo-osmotic shock, that confined to the smaller particles was resistant to this treatment. Resistance to hypo-osmotic conditions is not unique to LRH-containing particles, however. In this study, we noted that some of the particles containing NE also exhibited a similar property. If one accepts the hypothesis that the hypo-osmotically resistant compartment of neurotransmitters rep1969), then the resents their stable pool (WHITTAKER, stable pool of LRH is much larger than that of TRH. Moreover, the failure of O.lyo DOC to solubilize the LRH associated with the small particles further substantiates the existence of a stable pool of LRH. Nevertheless, 0.1% Triton X-100 solubilizes both pools of TRH and LRH. A consideration of the manner in which DOC solubilizes specific cellular constituents may provide a guide to the interpretation of these data in morphological terms. It has been suggested that DOC at a concentration of 0.26% solubilizes principally the contents of microsomal vesicles (ERNSTERet al., 1962). However, this property of DOC cannot be taken as an indication that TRH- and the large LRH-containing particles are microsomes, since the gradient fractions containing TRH and LRH do not possess the activity of NADPH-cytochrome c reductase (BARNEA et a/., 1975). On the other hand, peptide hormones such as parathyroid hormone are stored in vesicular structures from which the newly synthesized hormone, identical in structure to the old hormone, can be extracted preferentially by DOC (MacGregor et
483
Hypothalamic particles containing LRH and TRH
- 240
- 200
- 160
c
0
0
-120
z
L
a2
n
a cn
- 80
- 40
GRADIENT FRACTION
c,---032 0 4
,
1-1
14032 0 4
1.4 I
SUCROSE CONCENTRATION ( M I
FIG. 8. Etrect of DOC o n the gradient profiles of LRH and TRH. See Fig. 5 for details.
ol., 1973). Synaptic vesicles are assembled and trans- mic subcellular particles thought not to be synapto& KARAVOLAS, 1975). Moreover, homoported along the smooth endoplasmic reticulum somes (TABER which appears to be a continuous system extending genates of glial cells contain particles which have the from the Golgi apparatus to the presynaptic nerve same isopycnic banding density on sucrose as synapet a/., 1971). Therefore, the populaending (DROZ et a/., 1975). Furthermore, the sedimen- tosomes (COTMAN tation characteristics of fragments of the Golgi appar- tion of particles comprising the stable pool of LRH may be of glial origin. atus are similar to those of synaptosomes (FLEISCHER et al., 1969; CURD et al., 1974). Therefore, the possibility should be considered that some of the labile pool Ackriowledge,nenrs-We thank JUDYWAGERS and BRENDA MOST, of LRH and T R H represents newly synthesized pep- ROWLANDfor editorial assistance and CAROLINE MARGUERITE GUNDER, and tides as has been shown for acetylcholine (RICHTER ROBERTLIPSEY, SUE SHERWIN, GAYLE LOPICCOLO for technical assistance. This investiga& MARCHBANKS, 1971). The stable pool of acetylcholine has been identified tion was supported by a research grant (AM01237) from with synaptic vesicles which have an isopycnic band- the National Institute of Arthritis, Metabolism. and Digestive Diseases. ing density of approx 0.4 M-SUCTOSe (WHITTAKER, 1973). The T R H stable pool appears to have similar sedimentation characteristics (BARNEAet ul., 1975), REFERENCES but the stable pool of LRH bands at 1.27 M-sucrose, obviating an identity with synaptic vesicles. Therefore, BAKER B. L., DERMOUY W. C. & REEL J. R. (1975) Endoother explanations should be considered. For crinology 97. 125-135. example, the stable pool of LRH may consist of BALDESSARINI R. J. & VOGT M. (1971) J. Neurochrm. 18, synaptosomes which have different membrane charac951-962. A., BEN-JONATHAS N.. COLSTON C.. JOHNSTON teristics than do those containing TRH. or that the BARNEA I. M. & PORTER 3. C . (1975)Proc. mmi. .4cud. Sci.. U.S.A. mode of binding of LRH in the synaptosomes may 72, 3153- 3157. be different from that of classical neurotransmitters BARRY J., DUBOIS M. P. & CARETTE B. (1974) Endocrinoor TRH. logy 95, 1416-1423. An alternative interpretation includes the possiBEU-JONATHANN. & PORTERJ. C. Endocrinolog!. (In bility that these LRH-containing particles are not press). synaptosomes. It has been demonstrated by immuno- BROWSSTTINM. J., PALKOVITS M.. SAAVEDRA J. M.. BASIRI histochemical techniques that LRH is localized in R. M. & UTIGERR. D. (1974) Science 185, 267-269. et COTMAN neuronal as well as ependymal cells (ZIMMERMAN C.. HERSCHMAS H. & TAYLOR D. (1971)J. 'Veuroh i d . 2, 169-180. ul., 1974). In addition, a substance stimulating pituitary LH release has been extracted from hypothala- Df DWE C. (1971) J . Cell B i d 50, 20W55D.
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AYALLABARNEA. NIRABEN-JONATHAN and JOHN C. PORTER
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