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.     

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.     

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. III. EFFECT OF PROLACTIN ON THE HYDROXYLATION OF PROTOCOLLAGEN-PROLINE OF THE TAIL FIN

The fact that bovine prolactin stimulates the collagen synthesis of the tadpole tail fin was confirmed by the present experiments, in which specific radioactivity of ¹⁴C-hydroxyproline hydroxylated from the incorporated ¹⁴C-proline was measured by the method different from that of the previous report (Y oshizato and Y asumasu , 1970). Stimulatory effect of prolactin on the incorporation of ¹⁴C-proline into the collagen fraction did not disappear even under the conditions, where the activity of protocollagen-proline hydroxylase was inhibited by α,α'-dipyridyl. In fact prolactin had not a significant effect on the activity of the emzyme. It, therefore, is concluded that the hormone promotes the incorporation of ¹⁴C-proline into collagen without affecting the hydroxylation step of the protein synthesis.     

Gibberellic acid-stimulated and auxin-stimulated cell growth in relation to RNA synthesis, protein synthesis and development in the wheat coleoptile

Young excised coleoptiles from dark grown wheat have their cell growth promoted by gibberellic acid (GA₃), while sections from older coleoptiles have their cell growth promoted by auxin. The GA₃ response has a much longer lag period than that of auxin. Neither GA₃ nor auxin has any effect on ¹⁴C-leucine and ¹⁴C-uridine incorporation and uptake after 1 h, indicating that the lag in growth stimulation following GA₃ application is not associated with changes in protein or RNA synthesis. Following a 6 h incubation there are small increases in ¹⁴C-leucine and ¹⁴C-uridine incorporation in response to both GA₃ and auxin, and in the case of auxin this is associated with increased uptake. Studies on protein and RNA turnover using pulse-chase experiments have shown that both GA₃ and auxin have no effect on protein and RNA stability. There are, however, developmental changes in RNA and protein synthesis that should be considered in any explanation of the mechanism of action of these hormones on cell growth. Young GA₃-sensitive tissue has high rates of RNA synthesis and low protein and RNA turnover, while auxin-sensitive tissue has low rates of RNA synthesis, slightly higher rates of RNA turnover and much higher rates of protein turnover. The evidence overall favours more effective utilisation by GA₃ and auxin of a basal control level of RNA and protein synthesis and turnover in coleoptile tissue.     

Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

Abstract The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like ³H-thymidine ¹⁴C-uracil, ¹⁴C-leucine and ¹⁴C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

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.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

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.     

INCREASE IN THE RELATIVE RATE OF SYNTHESIS OF DOPAMINE-β-HYDROXYLASE IN IN THE NUCLEUS LOCUS COERULEUS ELICITED BY RESERPINE

Abstract— Three days following a single injection of reserpine (10 mg/kg, i.p.) the activity and amount of dopamine-β-hydroxylase (DBH) are increased nearly 2-fold in the noradrenergic cell bodies of the nucleus locus coeruleus of rat. To determine if this increased accumulation of DBH is due to an increased rate of enzyme synthesis, [³H]amino acids were infused into the IVth ventricle of reserpine-and saline-injected rats. This method was 35 times more effective than intracisternal infusion and 600 times more effective than intravenous infusion. DBH protein was isolated from the locus coeruleus by immunoprecipitation and SDS-electrophoresis. These steps proved crucial for the complete isolation of DBH from other labelled proteins. Indeed, only 10–15% of the immunoprecipitate was finally identified as labelled DBH protein. The rate of incorporation of [³H]leucine into DBH protein of locus coeruleus was increased to 181%, of control following reserpine, whereas that into TCA-precipitable protein was unchanged. A similar result was obtained using [³H]lysine. In contrast, the apparent half-life of the enzyme did not change following reserpine. The relative rate of synthesis of DBH ([³H]DBH/³H-total protein), denoting selectivity of response, was increased in the locus coeruleus of reserpine-treated rats to 154% of control ( P < 0.01). These findings indicate that increased synthesis accounts for the observed increase in DBH protein in the locus coeruleus following reserpine administration.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.