EFFECTS OF THYROXINE AND PROLACTIN ON COLLAGEN BREAKDOWN IN THE THIGH BONE AND TAIL FIN OF THE RANA CATESBEIANA TADPOLE

Injection of ¹⁴C-proline into the tadpole causes labeling of protein in the collagen fraction of the thigh bone and tail fin. The radioactivity of the ¹⁴C-hydroxyproline residue is about 26% of the total radioactivity in the ¹⁴C-labeled protein of the collagen fraction in the thigh bone as well as in the tail fin. In ¹⁴C-proline-loaded tadpoles into which prolactin has been injected, the radioactivity in the collagen fraction in these tissues is markedly higher than that in control animals. In thyroxine-treated tadpoles, the ¹⁴C-radioactivity of the collagen fraction in the thigh bone is always higher than that of the controls, but it is markedly low in the tail fin. During the incubation of thigh bone and tail fin isolated from ¹⁴C-proline-loaded tadpoles, low molecular weight materials containing ¹⁴C-hydroxyproline are released from the ¹⁴C-labeled protein of these tissues. The rate of ¹⁴C-hydroxyproline release, which represents the rate of collagen breakdown, is higher in thigh bone and tail fin isolated from thyroxine-treated tadpoles and is markedly lower in these tissues isolated from prolactin-treated tadpoles than in those isolated from controls. In these tissues, the high rate of collagen breakdown in thyroxine-treated tadpoles is reduced by prolactin injection.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. IV. EFFECT OF PROLACTIN ON THE METABOLIC FATE OF HYALURONIC ACID, COLLAGEN AND RNA WITH A SPECIAL REFERENCE TO CATABOLIC PROCESS

In the case of hyaluronic acid and collagen, treatment of tadpoles with bovine prolactin produced remarkable increase in the content of the two metabolites in the tail fin in accord with the stimulatory effect of the hormone on the incorporation of labeled precursors into hyaluronic acid and collagen. RNA content of the tissue was increased appreciably by the prolactin treatment, whereas the hormone had no effect on the incorporation of ¹⁴C-uridine into RNA. Prolactin had inhibitory effect on the activities of hyaluronidase and RNase of the tail fin. The experiments on the protein catabolism also revealed that prolactin inhibited the process of protein turn-over in the tissue.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. V. STIMULATORY EFFECT OF PROLACTIN ON THE INCORPORATION OF 3H-THYMIDINE INTO DNA OF THE TADPOLE TAIL FIN

DNA content of the tadpole tail fin was increased appreciably by the treatment of animals with bovine prolactin. Incorporation of ³H-thymidine into DNA in the hormone-treated tail fins was remarkably stimulated as compared to the control ones. From the experiment on the effect of prolactin on the DNase activity, it is concluded that prolactin not only stimulates the synthetic parthway, but also suppresses significantly the catabolic one of the DNA metabolism.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. I. STIMULATORY EFFECT OF PROLACTIN ON THE COLLAGEN SYNTHESIS OF THE TADPOLE TAIL FIN

Bovine prolactin stimulated the growth of connective tissues both in the tail fins and in other regions of the tadpole tail. Correlated with the morphological effect of the hormone on the tadpole tails, protein synthesis in tail fins was promoted about 2 times by prolactin. Experiments performed to determine the kind of protein, the synthesis of which was stimulated by prolactin, revealed that the hormone specifically enhanced collagen synthesis about 40 folds as compared to untreated animals.     

HORMONES CONTROLLING COLLAGEN SYNTHESIS DURING METAMORPHOSIS IN THE THIGH BONE OF THE TADPOLE OF RANA CATESBEIANA

The rate of ¹⁴C-proline incorporation into collagen in the thigh bone of the Rana catesbeiana tadpole was determined in vitro. Intraperitoneal injection of bovine prolactin caused an increase in the rate of collagen synthesis during the premetamorphic stages (stages 12–16) and the early metamorphic stage (stage 18), but it exerted no effect on collagen synthesis in the metamorphic stages (stages 20–25). On the other hand, injection of growth hormone stimulated the rate of collagen synthesis in the metamorphic stages and caused a slight increase in the premetamorphic stages. When a tadpole in the early premetamorphic stages (stages 12–14) was kept in 5 × 10₋₈ M thyroxine solution for several days, the rate of collagen synthesis became higher than that in the bone of the control animal. The rate of collagen synthesis was not enhanced by prolactin in the thyroxine-treated tadpole, but was stimulated by growth hormone, even when the thyroxine-treated animal remained in the premetamorphic stages. With the treatment of the tadpole by thyroxine, prolactin-sensitivity seems to be reduced, and growth hormone-sensitivity becomes apparent.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

EFFECT OF INSULIN ON THE INCORPORATION OF 14C OF RADIOACTIVE GLUCOSE INTO GLYCOGEN AND CARBON DIOXIDE IN CEREBRAL CORTICAL SLICES

Abstract— Slices from the cerebral cortices of normal and alloxan-diabetic rats were incubated with [U-²⁴C]glucose. When insulin was added to the incubation medium the incorporation of ¹⁴C into glycogen was significantly increased in both groups. Insulin did not appear to have any significant effect on the incorporation of ¹⁴C into carbon dioxide.     

EFFECT OF CHOLINE ON THE RATES OF SYNTHESIS and OF RELEASE OF ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

Abstract— Slices of electric organ of Torpedo marmorata were chopped and incubated in a saline-urea-sucrose medium. This preparation of minced tissue exhibited a relative enrichment in ACh and nerve endings, which was attributed to a loss of electroplaque cytoplasm. Electron microscopic controls showed nerve endings of normal morphology, some of them forming 'chaplets' separated from electro-plaques. Miniature endplate potentials were recorded on sealed fragments also present in this preparation. ACh levels remained unchanged during incubation periods as long as 19 h. The time course of the incorporation of [1-¹⁴C]acetate of [2-¹⁴C]pyruvate into ACh pools was studied. These incorporations were similarly affected by the choline added to the medium. In the presence of increasing choline concentrations (up to 10^-4 m ), the incorporation of [¹⁴C]acetate or [¹⁴C]pyruvate into ACh increased. They both diminished when choline was added above 10^-4M. The ACh content of the tissue was not affected by added choline. From the constancy of ACh levels in the presence of various choline concentrations and from the steady state of our preparation, we can conclude that the release of transmitter varied in parallel to the incorporation rate of the precursor of the acetyl moiety of ACh. This fact was also found using the efflux of [¹⁴C]acetate as an evaluation of ACh release. The values of release calculated by this method were in good agreement with those determined from the incorporations of acetate and pyruvate into ACh. It is suggested that the primary action of choline is on its high affinity carrier system. This triggers a secondary action on the ACh release mechanisms.     

THE EFFECT OF DENERVATION ON INCORPORATION OF 32P AND [3H]GLYCEROL BY THE MUSCLE MEMBRANE

Abstract— The incorporation in vivo of ³²P₁ was significantly increased in all glycerophosphatide of preparations of denervated muscle membrane in frogs. There was no increase in incorporation of ³²P₁ into sphingomyelin. Disuse induced by tenotomy did not significantly increase incorporation of ³²P₁ into phospholipids of the muscle membrane. The phospholipid content of muscle membranes remained unchanged as a result of denervation or tenotomy. Denervation produced an increase in the incorporation of [2-³H]glycerol into all glycerophosphatides in parallel with the increase in ³²P₁ incorporation. Although the stimulated incorporation of ³²P₁ was increased in the regions of the muscle membrane rich in endplates, the most marked effect was in the endplate-poor region where activity in phosphatidylserine was most markedly increased.     

EFFECT OF INSULIN ON LEVELS AND TURNOVER OF INTERMEDIATES OF BRAIN CARBOHYDRATE METABOLISM IN VIVO

Abstract— –The rates of incorporation of ¹⁴C from [U-^l4C]glucose into intermediary metabolites have been measured in rat brain in vivo. The time course of labelling of glycogen was similar to that of glutamate and of glucose, which were all maximally labelled between 20 and 40min, but different from lactate, which lost radioactivity rapidly after 20min. The extent of labelling of glycogen (d.p.m./ μ mol of glucose) was of the same order as that of glutamate at 20 and 40 min after injection of [¹⁴C]glucose. However, calculations of turnover rates showed that glutamate turns over some 8-10 times faster than glycogen. Insulin, intracisternally applied, produced after 4-5 h a 60 per cent increase in glucose-6-P and a 50 per cent increase in glycogen. There was no change in the levels of glucose, glutamate or lactate, nor in the activity or properties of the particulate and soluble hexokinase of the brain. The injection of insulin affected neither the glycogen nor glucose contents of skeletal muscle from the same animals. The effects of insulin on the incorporation of ^l4C into the metabolites contrasted with its effects on their levels. The specific activities of glycogen and glucose were unchanged and there was a slight but non-significant increase in the specific activity of glutamate. The time course of incorporation into lactate was unaffected up to 20 min, but a significant delay in the loss of ¹⁴C after 20 min occurred as a result of the insulin injection. At 40 min, the specific activity of cerebral lactate was 60 per cent higher in insulin-treated animals than in control animals. The results are interpreted in terms of an effect of insulin on glucose uptake to the brain, with possibly an additional effect on a subsequent stage in metabolism, which involves lactate.     

CITRATE AS THE PRECURSOR OF THE ACETYL MOIETY OF ACETYLCHOLINE

Abstract— Rat brain cortex slices were incubated with glucose labeled with either ³H or ¹⁴C in the 6-position. The ³H/¹⁴C ratios and the incorporation of radioactivity into lactate, citrate, malate and acetylcholine were determined. While the ³H/¹⁴C ratio of lactate was close to that of glucose, the ratios in the acetyl moiety of acetylcholine and the acetyl (C-4,5) portion of citrate decreased in a similar proportion. This was interpreted as indirect evidence for the participation of citrate as a precursor to the acetyl moiety of acetylcholine. Two inhibitors of the citrate cleavage pathway: n -butylmalonate, an inhibitor of citrate transport and (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase were studied for their effect on acetylcholine synthesis. N -butylmalonate (10 mM) and (-)-hydroxycitrate (7.5 mM) led to a decrease in the per cent of ¹⁴C recovered as acetylcholine. In each instance the ³H/¹⁴C ratio in acetylcholine was higher in the presence of inhibitor while the corresponding ratios in lactate and citrate (C-4.5) remained unchanged. From the results, it is suggested that citrate is involved in the transport mechanism of acetyl units from its site of synthesis in mitochondria to the site of acetylcholine synthesis in the cytosol.     

THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

Conversion of sucrose to starch in the developing Pennisetum typhoides grain

Developing grains of pearl millet ( Pennisetum typhoides Burm. S & H cv. PIB 155) were sampled and analyzed for starch and its free-sugar precursors. The activities of invertase, sucrose-ADP (UDP) glucosyl transferase and of α-amylase and β-amylase in relation to the rate of starch accumulation in the developing grain were assayed. By culturing detached ears, the incorporation of ¹⁴C from free sugar precursors to starch was studied. The starch content gradually increased until grain maturity. The rate of starch accumulation was maximum around 12 days after anthesis. Around this period, the activities of sucrose-ADP(UDP) glucosyl transferase and α-amylase, β-amylase were also at a peak. Invertase activity was high during the early period of grain development but gradually declined as the grains matured. In the most actively metabolising milky grains, incorporation of ¹⁴C from [¹⁴C]-sugars to starch was maximum in the mid mid-milky grains. Addition of 20 m M K⁺ to the culture solution did not affect the incorporation of ¹⁴C from supplied sucrose to the free sugar pool and to the starch of the grain, but Mg²⁺ supply at 20 m M concentration lowered ¹⁴C incorporation from exogenous sucrose to grain free sugars, although the utilization of the latter for starch synthesis was enhanced.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.