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Biochemistry of leucocytes: Survival in vitro, phosphorus metabolism and phagocytosis
240
International Conference on Radioisotopes in Scientific Research
159 Studies on the Regulation of Red Cell Production Aided in Isotopes. J. H.
LAWRENCE, D. C. VAN DYKE, J. F. GARClA, and A. N. CONTOPOULOS, LTniversitv of California, Berkeley, U.S.A. Radioactive isotopes have provided i m p o r t a n t means for learning more a b o u t the n a t u r e of the physiological mechanisms by which the body m a i n t a i n s a n a d e q u a t e supply of red cells in the circulation. T h e fact t h a t anoxia, such as t h a t found in a high altitude e n v i r o n m e n t or in certain types of h e a r t or p u l m o n a r y disease, gives rise to a polyeyt h a e m i a is well known. U n d e r such conditions the life span of the red cells is normal, a n d the polycythaemia is m a i n t a i n e d by a n increased rate of red cells production. A n experiment by REISSMANN in 1950 using parabiotic rats showed that w h e n one p a r a b i o n t was m a i n t a i n e d in a low oxygen atmosphere a n d the other in a n o r m a l oxygen environment, the red cell production was stimulated in b o t h animals, thus suggesting the existence of a h u m o r a l factor circulating in the blood which controlled red cell production. Since t h a t time, m a n y workers have reported the presence of a n erythropoietically active substance in the plasma a n d urine of animals m a d e anaemic by bleeding or p h e n y l h y d r a z i n e . T h e active factor has not been isolated, a n d its source is not known. Its presence has been detected by assay of plasma or urine samples from the anaemic animals in recipient hypophysectomized or n o r m a l animals. E r y t h r o poietic activity has b e e n measured by a variety of criteria, including increased red cell counts, h a e m o globin concentration, a n d haematocrits, all of which are subiect to possible error because of the possibility of h a e m o c o n c e n t r a t i o n or h a e m o d i l u a t i o n , as well as reticu]ocytosis, increased incorporation of Fe 59 by red cells, a n d increased red cell volume. T h e measurem e n t of red cell volume is p r o b a b l y the most reliable method, b u t it is time consuming. W e are now using Fe 59 incorporation as a r a p i d screening assay, with periodic checks of results by m e a s u r e m e n t of red cell volume. T h e site of p r o d u c t i o n of the erythropoietic f a c t o r - or factors, since there m a y be more t h a n o n e - - h a s not b e e n established. W e have found a n erythropoietic factor in pituitary extracts which seems to be allied chemically to A C T H , b u t the biological a n d chemical evidence suggests the presence of a distinct erythropoietic hormone. O t h e r workers have reported t h a t hypophysectomized animals m a d e a n a e m i c b y bleeding can produce a n erythropoietically active substance, b u t n e p h r e c t o m i z e d animals or animals which have b e e n ureter-ligated cannot, thus suggesting the kidney as a site of production. ~,Vork is now
in progress to d e t e r m i n e w h e t h e r n e p h r e c t o m y blocks production of a n erythropoietin directly by removal of the source or indirectly by toxic inhibition of p r o d u c t i o n of the substance elsewhere. Plasma erythropoietins have also been found in patients with polycythaemic a n d certain types of anaemia. W e have found t h a t extracts of plasma from patients with polycythaemia vera a n d secondary polycythaemia will increase the red cell volume of hypophyseetomized rats. Both native plasma a n d urine .from patients with aplastic a n a e m i a have also been shown to contain a n erythropoietically active substance w h e n assayed by the Fc 5'a incorporation method. U r i n e from the p a t i e n t with aplastie a n a e m i a whose plasma h a d showed the highest erythropoietic activity in one group of patients tested h a d sufficient activity to double the ineorporation of iron into h a e m o g l o b i n in normal rats w h e n given over a two-day period a n d to produce a significant increase in red cell volume in n o r m a l adult rats when given over a 14 day period.
160 Biochemistry of Leucocytes: Survival in Vitro, Phosphorus Metabolism and Phagocytosis. K . ZAKRZEWSKI, J . MALEC a n d W , BALASIEWICZ, I n s t i t u t e o f H a e m a t o logy, W a r s a w , P o l a n d . Leucocytes are the only m a m m a l i a n cell the metabolisrn of which can be studied in a long term experim e n t w i t h o u t previous mechanical or enzymatic p r e t r e a t m e n t . Such study has, hitherto, been h a m pered by the lack of a suitable m e t h o d for their isolation a n d cultivation in citro. I n this p a p e r a m e t h o d is described for isolation a n d cultivation in a'itro of a suspension of h u m a n n o r m a l circulatory leucocytes. T h e half-life of these cells has been found to be 36 hours. Phosphorus turnover was studied by a d d i n g pa2 to n u t r i e n t m e d i u m (as inorganic phosphate). Phosphorus incorporation into the ester phosphates of cytoplasm follows an a p p a r e n t first order reaction (kam ~ = 250 c p m / m g P / h ) , reaching equilibrium with the m e d i u m after a b o u t 48 hours. I n c o r p o r a t i o n of phosphorus into ribonucleic acids follows a n a p p a r e n t first order reaction (ko~ ~ -- 8 c p m / m g P / h ) , Specific activity of R N A - P reaches 3 4 per cent of the specific activity of ester phosphates. No i n c o r p a r a t i o n could be found in the desoxyribonucleic acid. Phagoeytosis is one of the most i m p o r t a n t nonspecific defences of m a m m a l i a n body against i n v a d i r g nficro-organisms. T h e new m e t h o d just described fi)r cultivation of circulatory leucocytes in ~,itro, makes possible a study of the biochemical m e c h a n i s m underlying phagocytosis.
241
Abstracts of Papers A suspension of heat-killed Staph. aureus has been added to a suspension of human leucocytes surviving in a nutrient medium containing Na2Hp3204. After incubation at 37°C, the cells were spun down, washed and homogenized in a sucrose solution. The nuclear fraction was purified by extraction with citric acid. Three fractions obtained, cytoplasmic, nuclear and "citric acid" were fractionated and phosphorus incorporation into phosphate esters and rihonucleic acid determined. It was found, that phagocytosis increases cytoplasmic R N A - P turnover more than tenfold; no increase in the ester phosphates turnover could be detected. Also, no activation of either nuclear or "citric acid" R N ~ was found.
162
161
C y t o e h e m i e a l S t u d i e s of N u c l e a r b o l i s m in B i o l o g i c a l S y s t e m s .
strated so far whether the original chromosome produces by a self-duplicating process a replica of itself from available new material ol whether the original chromosomal substance is equally distributed between the daughter nuclei. However, with the availability of specific precursors (like thymidine-2-C 14) of D N A and refinements in autoradiographic techniques it is now possible to selectively label particular components of chromosomes and follow the fate of the tagged atoms in successive mitotic cycles. Evidence is presented in support of the concept of a template process operating in chromosome synthesis and duplication.
MetaA. R.
GOPAL-AYENGAR, A t o m i c E n e r g y E s t a b l i s h ment and Indian Cancer Research Centre, Bombay, India. (1) Incorporation of adenine-8-C 14 in the oocytes of the newt and the somatic chromosomes of Vicia faba. With the aid of tract autoradiography using an electron sensitive liquid emulsion it has been possible to follow the intracellular incorporation of a labelled precursor like adenine-8-C 14 which is specific for nucleic acids. The incorporation was determined on the basis of topographical emission of ionising particles, in particular organelles of the cell. The balance of evidence points to the fact that in oocytes of the newt, the nucleolus incorporates adenine with marked rapidity in the R N A . However the fact that there is a high specific radioactivity in the nucleolus does not necessarily imply that the site of synthesis of R N A is centred in this organelle. For, in the actively dividing meristematic cells of Vicia faba there is a preferential uptake of the labelled adenine in the heterochromatic and nucleolar organising (SAT) regions of the long chromosomes. From this it would appear that the nucleoli act as a reservoir of R N A and that actual synthesis supervenes in the heterochromatic regions and SAT-loci of particular chromosomes.
(2) Chromosome synthesis and duplication in somatic chromosomes of Vicia faba and Luzula pu~purea using thymidine-2-C 14. In an attempt to understand the mechanics of chromosomes replication, cytologists have made many assumptions, the validity of which has not been tested on cytochemical ground until very recently. Although it has been known for long that during mitosis there is a doubling of chromosomes followed by an equipartition of chromosomal substance between the two daughter nuclei, no decisive technique has demon-
R o l e of V i t a m i n B12 in N u c l e i c A c i d M e t a b o l i s m . B. S. SCI~WEmEI~T, F. A. NAYDER, O . F. BATZER, a n d A. J . SmDI.ER, A m e r i c a n Meat Institute Foundation and Department of Biochemistry, University of Chicago, U.S.A. The vital role of vitamin BI~ in the nutrition of man and other animals and certain micro-organisms has been established for several years. While considerable progress has been made in elucidating the biochemical role of this vitamin, no specific enzyme system in which vitamin B12 functions as a co-factor has as yet been clearly defined. In the present work, the role of vitamin B12 in the metabolism of nucleic acids by the rat and Lactobaccillus leichmannii has been studied. Vitamin B12 has been shown to be required for deoxyribose synthesis by L. leichmannii based on metabolism studies with C 1~ labelled thymidine. The conversion of C 14 labelled cytidine to deoxyribosides by vitamin B12 deficient and supplemented rats is also being investigated. Certain purine bases plus a deoxynucleoside have been shown to be required to replace vitamin BI.~ for L. leichmannii. Guanine was the most active purine tested. In studies with Lactobacillus acidophilus (which requires a deoxyriboside not replaceable by vitamin B12) the requirement for various nucleic acid derivatives has been demonstrated. The addition of certain ribonucleotides appears to inhibit the utilization of deoxynucleotides by this organism. 163
l~tude du m 6 t a b o l i s m e d e s a c i d e s n u c l e i q u e s de l ' 6 p i t h e l i o m a a t y p i q u e du rat par l ' i n c o r p o r a t i o n du pa2. y . KHOUVINE, I n s t i t u t d e B i o l o g i c P h y s i c o c h i m i q u e , Paris, F r a n c e . Cette dtude a ~td faite in vivo et in vitro. In vivo: lorsque la tumeur est bien d6velopp~e, on
International Conference on Radioisotopes in Scientific Research
159 Studies on the Regulation of Red Cell Production Aided in Isotopes. J. H.
LAWRENCE, D. C. VAN DYKE, J. F. GARClA, and A. N. CONTOPOULOS, LTniversitv of California, Berkeley, U.S.A. Radioactive isotopes have provided i m p o r t a n t means for learning more a b o u t the n a t u r e of the physiological mechanisms by which the body m a i n t a i n s a n a d e q u a t e supply of red cells in the circulation. T h e fact t h a t anoxia, such as t h a t found in a high altitude e n v i r o n m e n t or in certain types of h e a r t or p u l m o n a r y disease, gives rise to a polyeyt h a e m i a is well known. U n d e r such conditions the life span of the red cells is normal, a n d the polycythaemia is m a i n t a i n e d by a n increased rate of red cells production. A n experiment by REISSMANN in 1950 using parabiotic rats showed that w h e n one p a r a b i o n t was m a i n t a i n e d in a low oxygen atmosphere a n d the other in a n o r m a l oxygen environment, the red cell production was stimulated in b o t h animals, thus suggesting the existence of a h u m o r a l factor circulating in the blood which controlled red cell production. Since t h a t time, m a n y workers have reported the presence of a n erythropoietically active substance in the plasma a n d urine of animals m a d e anaemic by bleeding or p h e n y l h y d r a z i n e . T h e active factor has not been isolated, a n d its source is not known. Its presence has been detected by assay of plasma or urine samples from the anaemic animals in recipient hypophysectomized or n o r m a l animals. E r y t h r o poietic activity has b e e n measured by a variety of criteria, including increased red cell counts, h a e m o globin concentration, a n d haematocrits, all of which are subiect to possible error because of the possibility of h a e m o c o n c e n t r a t i o n or h a e m o d i l u a t i o n , as well as reticu]ocytosis, increased incorporation of Fe 59 by red cells, a n d increased red cell volume. T h e measurem e n t of red cell volume is p r o b a b l y the most reliable method, b u t it is time consuming. W e are now using Fe 59 incorporation as a r a p i d screening assay, with periodic checks of results by m e a s u r e m e n t of red cell volume. T h e site of p r o d u c t i o n of the erythropoietic f a c t o r - or factors, since there m a y be more t h a n o n e - - h a s not b e e n established. W e have found a n erythropoietic factor in pituitary extracts which seems to be allied chemically to A C T H , b u t the biological a n d chemical evidence suggests the presence of a distinct erythropoietic hormone. O t h e r workers have reported t h a t hypophysectomized animals m a d e a n a e m i c b y bleeding can produce a n erythropoietically active substance, b u t n e p h r e c t o m i z e d animals or animals which have b e e n ureter-ligated cannot, thus suggesting the kidney as a site of production. ~,Vork is now
in progress to d e t e r m i n e w h e t h e r n e p h r e c t o m y blocks production of a n erythropoietin directly by removal of the source or indirectly by toxic inhibition of p r o d u c t i o n of the substance elsewhere. Plasma erythropoietins have also been found in patients with polycythaemic a n d certain types of anaemia. W e have found t h a t extracts of plasma from patients with polycythaemia vera a n d secondary polycythaemia will increase the red cell volume of hypophyseetomized rats. Both native plasma a n d urine .from patients with aplastic a n a e m i a have also been shown to contain a n erythropoietically active substance w h e n assayed by the Fc 5'a incorporation method. U r i n e from the p a t i e n t with aplastie a n a e m i a whose plasma h a d showed the highest erythropoietic activity in one group of patients tested h a d sufficient activity to double the ineorporation of iron into h a e m o g l o b i n in normal rats w h e n given over a two-day period a n d to produce a significant increase in red cell volume in n o r m a l adult rats when given over a 14 day period.
160 Biochemistry of Leucocytes: Survival in Vitro, Phosphorus Metabolism and Phagocytosis. K . ZAKRZEWSKI, J . MALEC a n d W , BALASIEWICZ, I n s t i t u t e o f H a e m a t o logy, W a r s a w , P o l a n d . Leucocytes are the only m a m m a l i a n cell the metabolisrn of which can be studied in a long term experim e n t w i t h o u t previous mechanical or enzymatic p r e t r e a t m e n t . Such study has, hitherto, been h a m pered by the lack of a suitable m e t h o d for their isolation a n d cultivation in citro. I n this p a p e r a m e t h o d is described for isolation a n d cultivation in a'itro of a suspension of h u m a n n o r m a l circulatory leucocytes. T h e half-life of these cells has been found to be 36 hours. Phosphorus turnover was studied by a d d i n g pa2 to n u t r i e n t m e d i u m (as inorganic phosphate). Phosphorus incorporation into the ester phosphates of cytoplasm follows an a p p a r e n t first order reaction (kam ~ = 250 c p m / m g P / h ) , reaching equilibrium with the m e d i u m after a b o u t 48 hours. I n c o r p o r a t i o n of phosphorus into ribonucleic acids follows a n a p p a r e n t first order reaction (ko~ ~ -- 8 c p m / m g P / h ) , Specific activity of R N A - P reaches 3 4 per cent of the specific activity of ester phosphates. No i n c o r p a r a t i o n could be found in the desoxyribonucleic acid. Phagoeytosis is one of the most i m p o r t a n t nonspecific defences of m a m m a l i a n body against i n v a d i r g nficro-organisms. T h e new m e t h o d just described fi)r cultivation of circulatory leucocytes in ~,itro, makes possible a study of the biochemical m e c h a n i s m underlying phagocytosis.
241
Abstracts of Papers A suspension of heat-killed Staph. aureus has been added to a suspension of human leucocytes surviving in a nutrient medium containing Na2Hp3204. After incubation at 37°C, the cells were spun down, washed and homogenized in a sucrose solution. The nuclear fraction was purified by extraction with citric acid. Three fractions obtained, cytoplasmic, nuclear and "citric acid" were fractionated and phosphorus incorporation into phosphate esters and rihonucleic acid determined. It was found, that phagocytosis increases cytoplasmic R N A - P turnover more than tenfold; no increase in the ester phosphates turnover could be detected. Also, no activation of either nuclear or "citric acid" R N ~ was found.
162
161
C y t o e h e m i e a l S t u d i e s of N u c l e a r b o l i s m in B i o l o g i c a l S y s t e m s .
strated so far whether the original chromosome produces by a self-duplicating process a replica of itself from available new material ol whether the original chromosomal substance is equally distributed between the daughter nuclei. However, with the availability of specific precursors (like thymidine-2-C 14) of D N A and refinements in autoradiographic techniques it is now possible to selectively label particular components of chromosomes and follow the fate of the tagged atoms in successive mitotic cycles. Evidence is presented in support of the concept of a template process operating in chromosome synthesis and duplication.
MetaA. R.
GOPAL-AYENGAR, A t o m i c E n e r g y E s t a b l i s h ment and Indian Cancer Research Centre, Bombay, India. (1) Incorporation of adenine-8-C 14 in the oocytes of the newt and the somatic chromosomes of Vicia faba. With the aid of tract autoradiography using an electron sensitive liquid emulsion it has been possible to follow the intracellular incorporation of a labelled precursor like adenine-8-C 14 which is specific for nucleic acids. The incorporation was determined on the basis of topographical emission of ionising particles, in particular organelles of the cell. The balance of evidence points to the fact that in oocytes of the newt, the nucleolus incorporates adenine with marked rapidity in the R N A . However the fact that there is a high specific radioactivity in the nucleolus does not necessarily imply that the site of synthesis of R N A is centred in this organelle. For, in the actively dividing meristematic cells of Vicia faba there is a preferential uptake of the labelled adenine in the heterochromatic and nucleolar organising (SAT) regions of the long chromosomes. From this it would appear that the nucleoli act as a reservoir of R N A and that actual synthesis supervenes in the heterochromatic regions and SAT-loci of particular chromosomes.
(2) Chromosome synthesis and duplication in somatic chromosomes of Vicia faba and Luzula pu~purea using thymidine-2-C 14. In an attempt to understand the mechanics of chromosomes replication, cytologists have made many assumptions, the validity of which has not been tested on cytochemical ground until very recently. Although it has been known for long that during mitosis there is a doubling of chromosomes followed by an equipartition of chromosomal substance between the two daughter nuclei, no decisive technique has demon-
R o l e of V i t a m i n B12 in N u c l e i c A c i d M e t a b o l i s m . B. S. SCI~WEmEI~T, F. A. NAYDER, O . F. BATZER, a n d A. J . SmDI.ER, A m e r i c a n Meat Institute Foundation and Department of Biochemistry, University of Chicago, U.S.A. The vital role of vitamin BI~ in the nutrition of man and other animals and certain micro-organisms has been established for several years. While considerable progress has been made in elucidating the biochemical role of this vitamin, no specific enzyme system in which vitamin B12 functions as a co-factor has as yet been clearly defined. In the present work, the role of vitamin B12 in the metabolism of nucleic acids by the rat and Lactobaccillus leichmannii has been studied. Vitamin B12 has been shown to be required for deoxyribose synthesis by L. leichmannii based on metabolism studies with C 1~ labelled thymidine. The conversion of C 14 labelled cytidine to deoxyribosides by vitamin B12 deficient and supplemented rats is also being investigated. Certain purine bases plus a deoxynucleoside have been shown to be required to replace vitamin BI.~ for L. leichmannii. Guanine was the most active purine tested. In studies with Lactobacillus acidophilus (which requires a deoxyriboside not replaceable by vitamin B12) the requirement for various nucleic acid derivatives has been demonstrated. The addition of certain ribonucleotides appears to inhibit the utilization of deoxynucleotides by this organism. 163
l~tude du m 6 t a b o l i s m e d e s a c i d e s n u c l e i q u e s de l ' 6 p i t h e l i o m a a t y p i q u e du rat par l ' i n c o r p o r a t i o n du pa2. y . KHOUVINE, I n s t i t u t d e B i o l o g i c P h y s i c o c h i m i q u e , Paris, F r a n c e . Cette dtude a ~td faite in vivo et in vitro. In vivo: lorsque la tumeur est bien d6velopp~e, on