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Turnover of the sulphate groups in mast cells of young and adult rats
Gram reaction and plasmolytic effects in bacteria
659
REFERENCES 1. BISSET,
2. ~
K. A.,
J. gen. Microbial.
7, 233 (1952).
ibid. 13, 442 (1955).
3, HALE, C. M. F. and BISSET, K. A., ibid. 15, 423 (1956). 4. KNAYSI, G., Elements of bacterial cytology, pp. 159-162. 2nd Ed., New York, 1951. 5. LGHXIS, 1:. and SMITH, N. H., J. ugr. Research 23, 401 (1923).
TURNOVER
Comstock
OF THE SULPHATE GROUPS IN MAST YOUNG AND ADULT RATS QUANTITATIVE
AUTORADIOGRAPHIC
Pub.
Co., Ithaca,
CELLS
OF
STUDY
G. GUIDOTTI Istituto
di Patologia
Generule, University
Received
February
of Milan,
Italy
20, 1957
ACCORDING to previous investigations
radioactive sulphate is easily incorporated and in vitro in the sulphate groups of chondroitinsulphuric acid [3, 51 and of some other sulphomucopolysaccharides like heparin and precursors [7]. The injected radiosulphate was autoradiographically recovered in the mast cells of the rats’ skin by BostrGm ef al. [4] and in the mast cells of some experimental skin tumours by Asboe-Hansen [l, 21. Furthermore the uptake of SsS-labelled sodium sulphate by the cutaneous mast cells of young rats was studied by means of stripping film autoradiography [7]. This paper is concerned with the uptake of radioactive sulphate by the mast cells of the rats’ skin; experimental animals of different ages were chosen in order to determine both the intensity of incorporation and the rate of exchange of the SO, groups according to the body senescence. This investigation was carried out quantitatively by means of a modified autoradiographic technique. Twenty-seven rats, weighing about 70 g and 18 adult rats weighing about 220 g were given a single subcutaneous injection of %-labelled sodium sulphare (0.75 ,uC/g body Wt.). Three animals of the first group were killed at times after 15’, 45’, 90’, 3, 6, 12, 24, 48, 144 hours following the injection and three animals at the time of the second group were killed after 3, 6, 12, 24, 48, 144 hours following the injection. in vivo
Experimental
Cell Research 12
G. Guidotti
Fig. l.-Skin of young rat. Electron-track autoradiography. Emulsion Ilford G5, thickness 50~. Alcian 8 GK staining. 800 x , immersion. Animal killed 15’ after the injection of the radiosulphate. No electron track is emerging from the mast cells. Fig. 2 -Skin of young rat. Same details as for Fig. 1. Animal killed 6 hrs after the injection of the radiosulphate. Scvcral electron tracks, emerging from the mast cells, are here clearly identifiable.
The abdominal region was shaved; a skin specimen was removed, fixed in Carnoy’s fluid and embedded in paraffin. Sections 5,~ thick were mounted on gelatin subbed glass slides, deparaffinized, hydrated and stained for 15’ with a 0.5 per cent solution of alcian 8 GN in Walpole buffer, pH 2.5. The dried sections were coated with Ilford G5 photographic emulsion (gel form) to a thickness of 50,~. The plates were desiccated for 2 hrs at 20°C in a slow current of dry air. The exposure at 0°C lasted 40 hrs. Our routine processing was previously described [6]. Four sections for each animal including a total of 150 mast cells with related electron tracks were counted on the screen of a photographic microscope. All results were corrected according to the isotope decay and referred to the time of injection of the labelled sulphate. Autoradiographic pictures obtained by our technique are shown in Figs. 1 and 2. The results of the count of the tracks emerging from the cutaneous mast cells of the young and adult rats are reported in Figs. 3 and 4. In both of them the number of counted tracks for 100 mast cells, together with its standard error, has been indicated on the ordinate; the logarithm of the time intervals elapsed between the injection and the killing of the single groups of rats has been indicated on the abscissa. Our results show that the maximum of radioactivity is practically the same in the mast cells of either young or adult rats; however the incorporation rate of Experimental
Cell Research 12
Turnover of sulphafe groups in rat masf cells
661
the tracer appears to depend upon the age of the animals: as shown in Figs. 3 and 4 the maximum of radioactivity in the mast cells of young and adult rats was reached in about 6 and 24 hrs respectively. According to these observations the radiosulphate is easily incorporated in vivo into the heparin or into the heparin-like compounds present in the mast cells of the rats. 150.
loo-
50~
I 1 P
O15’
45’
90’
3h
6h
12h
24h
48h
144 h
3h
6h
12h
24h
48h
144h
Fig. 3 (left).-Young rats. The number of the tracks counted for 100 mast cells with its standard error is reported on the ordinate; the logarithm of the time intervals between the injection of the radiosulphate and the killing of the single groups of rats has been indicated on the abscissa. Fig. 4 (right).-Adult rats. Same details as for Fig. 3.
In the limits of our experience and, as far as the age of the experimental animals is concerned, these mast cells do not show significantly different exchange intensities. This lack of variability of the single cells to take up the sulphate groups could mean that no metabolic alterations bound to the ageing of the enzymatic systems of the protoplasm is brought about the senescence. However, since the exchange rate is higher in the younger animals, the slowing of the 9 uptake of the adult rats could be explained with the interference of some other causes like the state of vascularization of the skin, the membrane permeability or some undefined conditions bound to the whole animal body. Experiments are in progress in order to clarify this point. REFERENCES G., Cancer Research 13, 587 (1953). ibid. 14, 94 (1954). BOSTRGM, H., J. Bio!. Chem. 196, 477 (1952). BOSTRBM, H., ODEBLAD, E. and FRIBERG, U., Acfa Pafhol. Microbial. &and. DZIEWIATKOWSKI, D. D., J. Biof. Chem. 189, 187 (1951). GUIDOTTI, G. and LEVI-SETTI, R., Stain Technof. 31, 57 (1956). JORPES, E., ODEBLAD, E. and BOSTRBM, H., Acfa Haemafof. 9, 273 (1953).
1. ASBOE-HANSEN,
2. 3. 4. 5. 6. 7.
-
Experimental
32, 516 (1953).
Cell Research 12
659
REFERENCES 1. BISSET,
2. ~
K. A.,
J. gen. Microbial.
7, 233 (1952).
ibid. 13, 442 (1955).
3, HALE, C. M. F. and BISSET, K. A., ibid. 15, 423 (1956). 4. KNAYSI, G., Elements of bacterial cytology, pp. 159-162. 2nd Ed., New York, 1951. 5. LGHXIS, 1:. and SMITH, N. H., J. ugr. Research 23, 401 (1923).
TURNOVER
Comstock
OF THE SULPHATE GROUPS IN MAST YOUNG AND ADULT RATS QUANTITATIVE
AUTORADIOGRAPHIC
Pub.
Co., Ithaca,
CELLS
OF
STUDY
G. GUIDOTTI Istituto
di Patologia
Generule, University
Received
February
of Milan,
Italy
20, 1957
ACCORDING to previous investigations
radioactive sulphate is easily incorporated and in vitro in the sulphate groups of chondroitinsulphuric acid [3, 51 and of some other sulphomucopolysaccharides like heparin and precursors [7]. The injected radiosulphate was autoradiographically recovered in the mast cells of the rats’ skin by BostrGm ef al. [4] and in the mast cells of some experimental skin tumours by Asboe-Hansen [l, 21. Furthermore the uptake of SsS-labelled sodium sulphate by the cutaneous mast cells of young rats was studied by means of stripping film autoradiography [7]. This paper is concerned with the uptake of radioactive sulphate by the mast cells of the rats’ skin; experimental animals of different ages were chosen in order to determine both the intensity of incorporation and the rate of exchange of the SO, groups according to the body senescence. This investigation was carried out quantitatively by means of a modified autoradiographic technique. Twenty-seven rats, weighing about 70 g and 18 adult rats weighing about 220 g were given a single subcutaneous injection of %-labelled sodium sulphare (0.75 ,uC/g body Wt.). Three animals of the first group were killed at times after 15’, 45’, 90’, 3, 6, 12, 24, 48, 144 hours following the injection and three animals at the time of the second group were killed after 3, 6, 12, 24, 48, 144 hours following the injection. in vivo
Experimental
Cell Research 12
G. Guidotti
Fig. l.-Skin of young rat. Electron-track autoradiography. Emulsion Ilford G5, thickness 50~. Alcian 8 GK staining. 800 x , immersion. Animal killed 15’ after the injection of the radiosulphate. No electron track is emerging from the mast cells. Fig. 2 -Skin of young rat. Same details as for Fig. 1. Animal killed 6 hrs after the injection of the radiosulphate. Scvcral electron tracks, emerging from the mast cells, are here clearly identifiable.
The abdominal region was shaved; a skin specimen was removed, fixed in Carnoy’s fluid and embedded in paraffin. Sections 5,~ thick were mounted on gelatin subbed glass slides, deparaffinized, hydrated and stained for 15’ with a 0.5 per cent solution of alcian 8 GN in Walpole buffer, pH 2.5. The dried sections were coated with Ilford G5 photographic emulsion (gel form) to a thickness of 50,~. The plates were desiccated for 2 hrs at 20°C in a slow current of dry air. The exposure at 0°C lasted 40 hrs. Our routine processing was previously described [6]. Four sections for each animal including a total of 150 mast cells with related electron tracks were counted on the screen of a photographic microscope. All results were corrected according to the isotope decay and referred to the time of injection of the labelled sulphate. Autoradiographic pictures obtained by our technique are shown in Figs. 1 and 2. The results of the count of the tracks emerging from the cutaneous mast cells of the young and adult rats are reported in Figs. 3 and 4. In both of them the number of counted tracks for 100 mast cells, together with its standard error, has been indicated on the ordinate; the logarithm of the time intervals elapsed between the injection and the killing of the single groups of rats has been indicated on the abscissa. Our results show that the maximum of radioactivity is practically the same in the mast cells of either young or adult rats; however the incorporation rate of Experimental
Cell Research 12
Turnover of sulphafe groups in rat masf cells
661
the tracer appears to depend upon the age of the animals: as shown in Figs. 3 and 4 the maximum of radioactivity in the mast cells of young and adult rats was reached in about 6 and 24 hrs respectively. According to these observations the radiosulphate is easily incorporated in vivo into the heparin or into the heparin-like compounds present in the mast cells of the rats. 150.
loo-
50~
I 1 P
O15’
45’
90’
3h
6h
12h
24h
48h
144 h
3h
6h
12h
24h
48h
144h
Fig. 3 (left).-Young rats. The number of the tracks counted for 100 mast cells with its standard error is reported on the ordinate; the logarithm of the time intervals between the injection of the radiosulphate and the killing of the single groups of rats has been indicated on the abscissa. Fig. 4 (right).-Adult rats. Same details as for Fig. 3.
In the limits of our experience and, as far as the age of the experimental animals is concerned, these mast cells do not show significantly different exchange intensities. This lack of variability of the single cells to take up the sulphate groups could mean that no metabolic alterations bound to the ageing of the enzymatic systems of the protoplasm is brought about the senescence. However, since the exchange rate is higher in the younger animals, the slowing of the 9 uptake of the adult rats could be explained with the interference of some other causes like the state of vascularization of the skin, the membrane permeability or some undefined conditions bound to the whole animal body. Experiments are in progress in order to clarify this point. REFERENCES G., Cancer Research 13, 587 (1953). ibid. 14, 94 (1954). BOSTRGM, H., J. Bio!. Chem. 196, 477 (1952). BOSTRBM, H., ODEBLAD, E. and FRIBERG, U., Acfa Pafhol. Microbial. &and. DZIEWIATKOWSKI, D. D., J. Biof. Chem. 189, 187 (1951). GUIDOTTI, G. and LEVI-SETTI, R., Stain Technof. 31, 57 (1956). JORPES, E., ODEBLAD, E. and BOSTRBM, H., Acfa Haemafof. 9, 273 (1953).
1. ASBOE-HANSEN,
2. 3. 4. 5. 6. 7.
-
Experimental
32, 516 (1953).
Cell Research 12