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Subcellular distribution of catecholamines in dog spleen
Lüe Sciences Vol. 5, pp . 457-4b4, 1966 . Printed in Great Britain.
SüBGELLULAR DISTRIBUTION
OF
Pergamon Press Ltd.
GATECHOLAMINES
IN DOG SPLEEN . P . M . Laduron, W . P. De Potter and A . F. De Schaepdryver J .F . and G .Heymaas Institute of Pharmacology, University of Great Medical School, Ghent, Belgium . (Received 3 deauaxy 1966) Endogenous catecholaminee have been shown to be stored, at least in part, to specific granules, as~indicated by studies on various tissue homogenates, e .g . adrenal medulla (1 ), splenic nerves (2) and brain~(3-5). Few data are available, however, on the subcellular distribution of these amines in relationship to other cellular particles, such as mitochondria, lysosomes and microsomea . The present work was done in order to study the subcellular localization of both endogenous and infused noradrenaline in dog spleen with reference to other cellular components, by characterizing a population of subcellular particles using marker enzymes as described by de Duve et al . (6) . Materials and Methods Young mongrel dogs wéighing 6-9 kg were anesthetized with pentobarbitone (30 mg/kg, i. v . ) and heparinized (20 mg/kg, i . v . ) . Polyethylene catheters were inserted into the splenic artery and vein . The spleen was then removed and perfused with Hanks solution at a constant output (710 ml/min) and temperature (37° C) according to the procedure described by Thoenen et al . (7) . A total dose of 0 .5-1 .0 pC of dl-noradrenaline G14 (spec . act . 21 .9 ~C/~M) was infused into the eplenic artery over a 3 hours period . 457
459
CA IIP DOG SPLEEN
Yol . 5, No .
5
Uptake of dl-noradrenaline Gl ~ in the spleen ranged from ~0 to 60 ~ of the dose infused. Upoa completion of the perfusion the spleen w;s immersed in ice cool 0,26 M sucrose, decspsulated, cut to small pieces and homogenised with a Potter-Elvehjem (Teflon pestle) in 5 volumes of ice cold 0 .25 M
sucrose buffered with 10 -3 M Tris at pH T. ~ and containing 10 -4 M MgGlZ (STM) . The homogenate was sabjected to differential centrüugatton as described by de Duve et al . (6), adapted to dog spleen in order to obtain as Lrge an enrichment as possible for the various fractions . The nuclear fraction was obtained by a low speed centrüugation, i. e . 500 g for 10 min . , and the cytoplasmic eztract centrifuged successively at 7, T00 g for 10 min . , 30, 800 g for 25 min . and 100, 000 g (Model L Spinco Ultracantrifuge) for 60 min. , giving .three particulate fractions, L.e . two mttochondrial and one microsomal fraction . Each fraction was wabhed twice and the sapernatants pooled for the ne :t step . Thus a nuclear (N), mitochondríal (M), light mitochondrial (L), microsomal (P) and supernatant (S) fraction was obtained . After fractionation a sample of each fraction was used for enzyme assay and RNA estimation in order to assess the composition of the various fractions . The following markers were adopted : cytochrome ozidase for M, acid phosphatase for L and RNA for P . Cytochrome ozidase was assayed using the optical test according to Cooperstein and Lazarow (8) . Total acid phospbatase was determined with 0 .1 ~ Triton X-100 but using
~ -glycerophosphate as substrate, in a
final concentration of 0 .15 M. Kinetics and latency of enzyme activities wül be published in detail (q) . RNA was extracted with hot phenol according to Gocito and Laduron (10) and UV absorbance measured in a Zeies
CA ~ DOG SPLEiSA
Vol . 5, No . 5
X59
spectrophotometer . Nttrogen was determined by a micro-Kjeldahl procidnre. Radioactivity of noradrenaline and metabalttes was measured in a Pacitard Liquid Scintillator Spectrometer after chromatographic separation as des cribed previously (ll) . Endogenous noradrenaline was assayed fluorome trically (12) . Results In preliminary a:perimsnts it was found that the subcellular particles of dug spleen have a different sedimentation coefficient as compared with those of rat liver, using the scheme of differential centrifugation proposed by de Duve et al . (6) . By applying highly increased Integrated forces, ms :imal values of relative specific activity may be obtatùed for both cytochrome o:idase in the M fraction and acid phosphatase in the L fraction, as illustrated in Fig . 1 .
>n i0 !0 ~0 Prre .roo. d lold ~s o0w,
100
FIG. 1 . Distribution patterns of enaymes, RNA and total radioactivity in dog spleen perfused with dl-noradrenaline Gl~ .
a~so
ca n~ noG sPL~x
vol . 5, xo. 5
The ht fraction contains on an average 51 °~ of total cytochrome okidaee and the L fraction 23
~o
of total acid phosphatase, whereas the content of
the latter enzyme in the M and P fractions amounts to 12 % and 17 90 respectively . In the not perfueed ppleen, however, the L fraction was found to contain over 30
~o
of total acid phosphatase . In this case the acid phos-
phatase activity in the S fraction was lower than ~n the perfueed spleen, indicating that part of the bound acid phoephatase is released either during perfusion or during subsequent fractionation . This observation may be compared to the one reported by de Duve and Beaufay (13) in a rat liver lobe rendered completely ischaemic by ligation . The relative specific activity of RNA was found to be maximal in the P fraction, although 40 70 of total RNA occurred in the S fraction . The latter corresponds to S-RNA (soluble RNA), while the RNA in the P frac tion is R-RNA (ribosomal RNA) or high molecular weight RNA (14) . As further shown in Fig . 1 the pattern of distribution of total radioactivity after infusion of dl-noradrenaline Cl4 ie very similar, expressed in terms of relative specific activity, to the one found for acid phospbatase . In cytoplaemic extract 46 h. of total radioactivity was found in the particulate fraction against 54 % in the soluble fraction . The corresponding values for endogenous noradrenaline were of 77 7o in the particulate frac tion and 23 ~% in the soluble fraction . In the perfueed spleen experiments, however, an important part of the radioactivity found in the soluble fraction was due to the presence of noradrenaline metabolites,
essentially
3-methoxy-4-hydroxymandelic acid, of which traces only were found to occur in the particulate fraction (15) . The experimental observations reported in Fig . 2 indicate that both acid phosphatase and the labelled substances are simultaneously released
Vol .
5, No . 5
CA IN DOG SPLEIIP
461
into the soluble fraction in presence of the same concentration of Triton X-100, probably by an action on the particles or the membrane of the particles . ,° ~
t sa
' .
° +
__1 to
FIG. 2
Samples of L fraction corresponding to 0 .5 g of spleen tissue are incabated at 0°G for 60 min . in presence of different concentrations of Triton X-100 . After incubation the samples are centrifuged at 30,800 g for 25 min. Pellet and supernatant are assayed for acid pbosphatase and radioactivity . Total activities are determined in not centrüuged samples. +
: acid phosphabse : radioactivity
ero"
Cor~e.r~lnöon ot Trllon (q/n~l)
Finally, the release of radioactivity from the L fraction was studied upon thermal activation at 37 ° G . In these ezperiments the amount of labelled material released at 37 ° C into the soluble fraction increased gradually to a plateau reached after 40 min ., whereas no release occurred at 0 ° G. Adding Triton X-100 in a final concentration of 10
-4
g/ml after
incubation and just before centrifugation did not increase radioactivity in the soluble fraction to more than 50 ~ of total . It seems therefore that adsorption alone cannot explain . why 50 ~° of the radioactivity remains to bound form in the granules . These data rather suggest that part of the granular content is labile while another part is present in a more stable form . These experimental obeervatia~ne are illnetrated in Fig. i°
462
CA IN DCG APLEIIQ
Yol. 5, No . 5
rc s~a-sr
+o~
~o
~mw,
-F`IG . 3
Samples of L fractions corresponding to 0 .5 g of spleen tissue are incubated at 0' G and 37' C for different periods of time in STM medium as described in Materials and Methods . After incubation 4 .5 ml of ice cold medium contain nTg Triton X-lÓ~ln a final concentration of 10 -4 g/ml is added and the mi:tore centrifuged at 30,SOO .g for 25 min . Pellet and supernatant are assayed for radioactivity . Total activities are determined in not centrifuged samples . Discussion and Conclusions 1 . Our experimental observations indicate that in dog spleen both endogenous and perfused catecholamines occur, to a large a:tent, in the particulate fraction . The experiments in Which the influence of Triton X-100 and of therma~ activation on the release of catecholamines Were studied produce additional evidence for this point of view . 2 . Both acid phosphatase and Lbelled compounds Were found to present a similar distribution pattern With a high reùtive epeeific acitivity in the L fraction . This and bhe fact that they are simultaneously released in presence of the same concentration of Triton X-100 does not necessarily imply that they occur in the same particles . Density gradient studies have shown, on the contrary, that lysosomes and catecholamiaee granules can be distinctly düferentiated from each other (15) . These findings are in
voi . 5, xo . 5
cA n~ noG
fi~~
a~s3
agreement with recent observations reported by BLschko et al . for bovine adrenal medulla (16) . 3 . The incomplete release of granular catecholamines upon. thermal activation of the L fraction suggests that the amine is present in the granules partly in labile form and partly to stable form,
the latter one being not
released . 4 . Finally,
some indications In this paper point to the presence,
spleen, of lysosomes, lases (9) . However,
to dog
a group of subcellular particles containing hydro-
homogenisation of spleen tissue presents many diffi-
anltles as reveáled by the reLtively high concentration of acid phosphateas in the supernatant (17) . Accordingly, the Weal procedure vhonld provide a sufficient degree of homogenisation without disrupting the particles . Summary Subcellular fractionation of dog spleen by differential centrifugation has shown both endogenous and infused labelled noradrenaline to occur to a large eztent in particle-bound form, the subcelluLr distribution of which is very similar to the pattern of distribution of acid phosphatase . Thermal activation a:periments suggest that this particle-bound amine occurs partly to labile form and partly In a more stable form . There are indications pointing to the presence of lysosomes in dog spleen . A cknowledg monts This work was supported by a grant from the Fund for Collective Fundamental Research, Belgium . The skilful technical assistance of Miss B .De Loore Is gratefully acknowledged .
464
CA IN DOG SPLEEN
Vol . 5, No . 5
R efeiences 1.
H .BLASCHKO and A .D .WELCH, Arch . exp . Path . Pharma3col . 219 .
2.
H . WEIL-MALHERBE and A .D .BONE, Nature , 180, 1050 (1957) .
3.
E .DE ROBERTIS, Histophysialogy of synapses and Neurosecretion,
4.
V .P .WHITTAKER, in "Biogenie Amines " ed . by H ..E .HIMWICH and
5.
U . S. von EDLER and N .A .HILLARP, Nature , 177, 44 (1956) .
6.
C . de DUVE, B .PRESSMAN, R .GIANETTO, R .WATTIAUX and
7.
H .THOENEN, A .HURLIMANN and W .HAEFELY, Helv . $1}y~l . Acta ,
8.
S.J .000PERSTEIN and A .LAZAROW, J . Biol . Ghem . _l89 . 665 (1951) .
9.
W .P .DE POTTER and P .M .LADURON, Arch . int. Physiol . Biochem .
10 .
G .COCITO and P .LADURON, Anal . Biochem. T, 429 (1964) .
11 .
W .DE POTTER, Z .M .BACQ, G .CRIEL, A .F .DE SCHAEPDRYVER
12 .
A .F .DE SCHAEPDRYVER, Arch . -int. Pharma c~n . 115, 233 (1958) .
13 .
G . de DUVE and H .BEAUFAY, Biochem . J . 73, 610 (1959) .
14 .
G . COCITO, Biochemical properties and metabolism of the nucleic
1 7 (1953) .
Pergamon Prees, Osford - London, 1964 .
W .A .HIMWICH, Elsevier, Amsterdam, p. 90 (1964) .
F .APPELMANS, Biochem. J . , 60, 604 (1955) . 21, 1T (1963) .
in prees .
and J .RENSON, Biochem. Pharir~acol. _12, 661 (1963) .
acids of viruses, cells aad virus-infected cells , Thesis, Foateyn, L,ouvsin, 1963 .
15 .
P .M .LADURON, W .P .DE POTTER and A .F .DE SCHAEPDRYVER,
16 .
H .BLASCHKO, A .D .SMITH and H .WINKLER, Proc . Physiol . Soc .
17 .
C . de DUVE . , in "Lyeosomee", A Ciba Foundation Symposium, J . & A . Churchill, London, p . 5 (1963) .
Biochem . Pharmacol, in press .
Lond . , 16 - 17 July 1965, C .12 .
SüBGELLULAR DISTRIBUTION
OF
Pergamon Press Ltd.
GATECHOLAMINES
IN DOG SPLEEN . P . M . Laduron, W . P. De Potter and A . F. De Schaepdryver J .F . and G .Heymaas Institute of Pharmacology, University of Great Medical School, Ghent, Belgium . (Received 3 deauaxy 1966) Endogenous catecholaminee have been shown to be stored, at least in part, to specific granules, as~indicated by studies on various tissue homogenates, e .g . adrenal medulla (1 ), splenic nerves (2) and brain~(3-5). Few data are available, however, on the subcellular distribution of these amines in relationship to other cellular particles, such as mitochondria, lysosomes and microsomea . The present work was done in order to study the subcellular localization of both endogenous and infused noradrenaline in dog spleen with reference to other cellular components, by characterizing a population of subcellular particles using marker enzymes as described by de Duve et al . (6) . Materials and Methods Young mongrel dogs wéighing 6-9 kg were anesthetized with pentobarbitone (30 mg/kg, i. v . ) and heparinized (20 mg/kg, i . v . ) . Polyethylene catheters were inserted into the splenic artery and vein . The spleen was then removed and perfused with Hanks solution at a constant output (710 ml/min) and temperature (37° C) according to the procedure described by Thoenen et al . (7) . A total dose of 0 .5-1 .0 pC of dl-noradrenaline G14 (spec . act . 21 .9 ~C/~M) was infused into the eplenic artery over a 3 hours period . 457
459
CA IIP DOG SPLEEN
Yol . 5, No .
5
Uptake of dl-noradrenaline Gl ~ in the spleen ranged from ~0 to 60 ~ of the dose infused. Upoa completion of the perfusion the spleen w;s immersed in ice cool 0,26 M sucrose, decspsulated, cut to small pieces and homogenised with a Potter-Elvehjem (Teflon pestle) in 5 volumes of ice cold 0 .25 M
sucrose buffered with 10 -3 M Tris at pH T. ~ and containing 10 -4 M MgGlZ (STM) . The homogenate was sabjected to differential centrüugatton as described by de Duve et al . (6), adapted to dog spleen in order to obtain as Lrge an enrichment as possible for the various fractions . The nuclear fraction was obtained by a low speed centrüugation, i. e . 500 g for 10 min . , and the cytoplasmic eztract centrifuged successively at 7, T00 g for 10 min . , 30, 800 g for 25 min . and 100, 000 g (Model L Spinco Ultracantrifuge) for 60 min. , giving .three particulate fractions, L.e . two mttochondrial and one microsomal fraction . Each fraction was wabhed twice and the sapernatants pooled for the ne :t step . Thus a nuclear (N), mitochondríal (M), light mitochondrial (L), microsomal (P) and supernatant (S) fraction was obtained . After fractionation a sample of each fraction was used for enzyme assay and RNA estimation in order to assess the composition of the various fractions . The following markers were adopted : cytochrome ozidase for M, acid phosphatase for L and RNA for P . Cytochrome ozidase was assayed using the optical test according to Cooperstein and Lazarow (8) . Total acid phospbatase was determined with 0 .1 ~ Triton X-100 but using
~ -glycerophosphate as substrate, in a
final concentration of 0 .15 M. Kinetics and latency of enzyme activities wül be published in detail (q) . RNA was extracted with hot phenol according to Gocito and Laduron (10) and UV absorbance measured in a Zeies
CA ~ DOG SPLEiSA
Vol . 5, No . 5
X59
spectrophotometer . Nttrogen was determined by a micro-Kjeldahl procidnre. Radioactivity of noradrenaline and metabalttes was measured in a Pacitard Liquid Scintillator Spectrometer after chromatographic separation as des cribed previously (ll) . Endogenous noradrenaline was assayed fluorome trically (12) . Results In preliminary a:perimsnts it was found that the subcellular particles of dug spleen have a different sedimentation coefficient as compared with those of rat liver, using the scheme of differential centrifugation proposed by de Duve et al . (6) . By applying highly increased Integrated forces, ms :imal values of relative specific activity may be obtatùed for both cytochrome o:idase in the M fraction and acid phosphatase in the L fraction, as illustrated in Fig . 1 .
>n i0 !0 ~0 Prre .roo. d lold ~s o0w,
100
FIG. 1 . Distribution patterns of enaymes, RNA and total radioactivity in dog spleen perfused with dl-noradrenaline Gl~ .
a~so
ca n~ noG sPL~x
vol . 5, xo. 5
The ht fraction contains on an average 51 °~ of total cytochrome okidaee and the L fraction 23
~o
of total acid phosphatase, whereas the content of
the latter enzyme in the M and P fractions amounts to 12 % and 17 90 respectively . In the not perfueed ppleen, however, the L fraction was found to contain over 30
~o
of total acid phosphatase . In this case the acid phos-
phatase activity in the S fraction was lower than ~n the perfueed spleen, indicating that part of the bound acid phoephatase is released either during perfusion or during subsequent fractionation . This observation may be compared to the one reported by de Duve and Beaufay (13) in a rat liver lobe rendered completely ischaemic by ligation . The relative specific activity of RNA was found to be maximal in the P fraction, although 40 70 of total RNA occurred in the S fraction . The latter corresponds to S-RNA (soluble RNA), while the RNA in the P frac tion is R-RNA (ribosomal RNA) or high molecular weight RNA (14) . As further shown in Fig . 1 the pattern of distribution of total radioactivity after infusion of dl-noradrenaline Cl4 ie very similar, expressed in terms of relative specific activity, to the one found for acid phospbatase . In cytoplaemic extract 46 h. of total radioactivity was found in the particulate fraction against 54 % in the soluble fraction . The corresponding values for endogenous noradrenaline were of 77 7o in the particulate frac tion and 23 ~% in the soluble fraction . In the perfueed spleen experiments, however, an important part of the radioactivity found in the soluble fraction was due to the presence of noradrenaline metabolites,
essentially
3-methoxy-4-hydroxymandelic acid, of which traces only were found to occur in the particulate fraction (15) . The experimental observations reported in Fig . 2 indicate that both acid phosphatase and the labelled substances are simultaneously released
Vol .
5, No . 5
CA IN DOG SPLEIIP
461
into the soluble fraction in presence of the same concentration of Triton X-100, probably by an action on the particles or the membrane of the particles . ,° ~
t sa
' .
° +
__1 to
FIG. 2
Samples of L fraction corresponding to 0 .5 g of spleen tissue are incabated at 0°G for 60 min . in presence of different concentrations of Triton X-100 . After incubation the samples are centrifuged at 30,800 g for 25 min. Pellet and supernatant are assayed for acid pbosphatase and radioactivity . Total activities are determined in not centrüuged samples. +
: acid phosphabse : radioactivity
ero"
Cor~e.r~lnöon ot Trllon (q/n~l)
Finally, the release of radioactivity from the L fraction was studied upon thermal activation at 37 ° G . In these ezperiments the amount of labelled material released at 37 ° C into the soluble fraction increased gradually to a plateau reached after 40 min ., whereas no release occurred at 0 ° G. Adding Triton X-100 in a final concentration of 10
-4
g/ml after
incubation and just before centrifugation did not increase radioactivity in the soluble fraction to more than 50 ~ of total . It seems therefore that adsorption alone cannot explain . why 50 ~° of the radioactivity remains to bound form in the granules . These data rather suggest that part of the granular content is labile while another part is present in a more stable form . These experimental obeervatia~ne are illnetrated in Fig. i°
462
CA IN DCG APLEIIQ
Yol. 5, No . 5
rc s~a-sr
+o~
~o
~mw,
-F`IG . 3
Samples of L fractions corresponding to 0 .5 g of spleen tissue are incubated at 0' G and 37' C for different periods of time in STM medium as described in Materials and Methods . After incubation 4 .5 ml of ice cold medium contain nTg Triton X-lÓ~ln a final concentration of 10 -4 g/ml is added and the mi:tore centrifuged at 30,SOO .g for 25 min . Pellet and supernatant are assayed for radioactivity . Total activities are determined in not centrifuged samples . Discussion and Conclusions 1 . Our experimental observations indicate that in dog spleen both endogenous and perfused catecholamines occur, to a large a:tent, in the particulate fraction . The experiments in Which the influence of Triton X-100 and of therma~ activation on the release of catecholamines Were studied produce additional evidence for this point of view . 2 . Both acid phosphatase and Lbelled compounds Were found to present a similar distribution pattern With a high reùtive epeeific acitivity in the L fraction . This and bhe fact that they are simultaneously released in presence of the same concentration of Triton X-100 does not necessarily imply that they occur in the same particles . Density gradient studies have shown, on the contrary, that lysosomes and catecholamiaee granules can be distinctly düferentiated from each other (15) . These findings are in
voi . 5, xo . 5
cA n~ noG
fi~~
a~s3
agreement with recent observations reported by BLschko et al . for bovine adrenal medulla (16) . 3 . The incomplete release of granular catecholamines upon. thermal activation of the L fraction suggests that the amine is present in the granules partly in labile form and partly to stable form,
the latter one being not
released . 4 . Finally,
some indications In this paper point to the presence,
spleen, of lysosomes, lases (9) . However,
to dog
a group of subcellular particles containing hydro-
homogenisation of spleen tissue presents many diffi-
anltles as reveáled by the reLtively high concentration of acid phosphateas in the supernatant (17) . Accordingly, the Weal procedure vhonld provide a sufficient degree of homogenisation without disrupting the particles . Summary Subcellular fractionation of dog spleen by differential centrifugation has shown both endogenous and infused labelled noradrenaline to occur to a large eztent in particle-bound form, the subcelluLr distribution of which is very similar to the pattern of distribution of acid phosphatase . Thermal activation a:periments suggest that this particle-bound amine occurs partly to labile form and partly In a more stable form . There are indications pointing to the presence of lysosomes in dog spleen . A cknowledg monts This work was supported by a grant from the Fund for Collective Fundamental Research, Belgium . The skilful technical assistance of Miss B .De Loore Is gratefully acknowledged .
464
CA IN DOG SPLEEN
Vol . 5, No . 5
R efeiences 1.
H .BLASCHKO and A .D .WELCH, Arch . exp . Path . Pharma3col . 219 .
2.
H . WEIL-MALHERBE and A .D .BONE, Nature , 180, 1050 (1957) .
3.
E .DE ROBERTIS, Histophysialogy of synapses and Neurosecretion,
4.
V .P .WHITTAKER, in "Biogenie Amines " ed . by H ..E .HIMWICH and
5.
U . S. von EDLER and N .A .HILLARP, Nature , 177, 44 (1956) .
6.
C . de DUVE, B .PRESSMAN, R .GIANETTO, R .WATTIAUX and
7.
H .THOENEN, A .HURLIMANN and W .HAEFELY, Helv . $1}y~l . Acta ,
8.
S.J .000PERSTEIN and A .LAZAROW, J . Biol . Ghem . _l89 . 665 (1951) .
9.
W .P .DE POTTER and P .M .LADURON, Arch . int. Physiol . Biochem .
10 .
G .COCITO and P .LADURON, Anal . Biochem. T, 429 (1964) .
11 .
W .DE POTTER, Z .M .BACQ, G .CRIEL, A .F .DE SCHAEPDRYVER
12 .
A .F .DE SCHAEPDRYVER, Arch . -int. Pharma c~n . 115, 233 (1958) .
13 .
G . de DUVE and H .BEAUFAY, Biochem . J . 73, 610 (1959) .
14 .
G . COCITO, Biochemical properties and metabolism of the nucleic
1 7 (1953) .
Pergamon Prees, Osford - London, 1964 .
W .A .HIMWICH, Elsevier, Amsterdam, p. 90 (1964) .
F .APPELMANS, Biochem. J . , 60, 604 (1955) . 21, 1T (1963) .
in prees .
and J .RENSON, Biochem. Pharir~acol. _12, 661 (1963) .
acids of viruses, cells aad virus-infected cells , Thesis, Foateyn, L,ouvsin, 1963 .
15 .
P .M .LADURON, W .P .DE POTTER and A .F .DE SCHAEPDRYVER,
16 .
H .BLASCHKO, A .D .SMITH and H .WINKLER, Proc . Physiol . Soc .
17 .
C . de DUVE . , in "Lyeosomee", A Ciba Foundation Symposium, J . & A . Churchill, London, p . 5 (1963) .
Biochem . Pharmacol, in press .
Lond . , 16 - 17 July 1965, C .12 .