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Thyroid hormone bioactivity is finely regulated at the cellular level by the peripheral iodothyronine deiodinases (D). The study of thyroid function in fish has been restricted mainly to teleosts, whereas the study and characterization of Ds have been overlooked in chondrichthyes. Here we report the cloning and operational characterization of both the native and the recombinant hepatic type 3 iodothyronine deiodinase in the tropical shark Chiloscyllium punctatum. Native and recombinant sD3 show identical catalytic activities: a strong preference for T3-inner-ring deiodination, a requirement for a high concentration of DTT, a sequential reaction mechanism, and resistance to PTU inhibition. The cloned cDNA contains 1298 nucleotides [excluding the poly(A) tail] and encodes a predicted protein of 259 amino acids. The triplet TGA coding for selenocysteine (Sec) is at position 123. The consensus selenocysteine insertion sequence (SECIS) was identified 228 bp upstream of the poly(A) tail and corresponds to form 2. The deduced amino acid sequence was 77% and 72% identical to other D3 cDNAs in fishes and other vertebrates, respectively. As in the case of other piscivore teleost species, shark expresses hepatic D3 through adulthood. This characteristic may be associated with the alimentary strategy in which the protection from an exogenous overload of thyroid hormones could be of physiological importance for thyroidal homeostasis.

Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T 2 , generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T 4 , T 3 , and 3,5-T 2 (0.1 μM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T 4 , T 3 , and 3,5-T 2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T 2 can affect both activity and transcription of hepatic deiodinases in teleosts.

We measured the activities of iodothyronine-activating (D1 and D2) and -inactivating (D3) deiodinases, as well as the circulating levels of their corresponding substrates and/or products, T4 ,T 3 ,a nd rT 3, in the developing rainbow trout. The study was conducted through 20 consecutive months and included fry, fingerlings, and mature animals from 14 to 88 weeks post-hatching (ph). Initially, blood concentration of active iodothyronines was low and tissue D3 activity was greater than the activity of D1 and D2. As fry developed into fingerlings, the activity of liver D2 increased, while that of skin D3 declined. The time course of these changes was directly (T3) or inversely (rT3 and D3) correlated to an increase in age, length, and body weight. The initial increment in both circulating T3 and hepatic D2 activity during the first spring (March-April; weeks 46–50 ph) coincided with the onset of gonadal differentiation, whereas the highest values of T3 and hepatic D1 and D2 (September–October; weeks 70–74 ph) occurred at the onset of sexual maturity. These results suggest that a hepatic supply of active T3 is temporally associated with gametogenesis. Abbreviations: ANS – 8-anilin-1-naphthalene sulphonic acid; DTT – Dithiothreitol; ph – post-hatching; T4 – thyroxine; T3 – triiodothyronine; rT3 – reverse triiodothyronine; T2 – diiodothyronine.

The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3′ untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.

In this study we analyzed whether corticosterone synthesis is involved in the regulation of adrenal gland type II deiodinase (AG-D2) activity during acute cold exposure. Two well-known inhibitors of steroidogenesis, aminoglutethimide (AGT) and metyrapone (MTP), were administered to male Wistar rats maintained either at room temperature or acutely exposed to cold (1 h at 4°C). AG-D2 activity was measured by the radioiodide release method, and corticosterone circulating levels were measured by competitive protein binding assay. Results show that resting corticosterone levels and AG-D2 activity were lower in both AGT-and MTP-treated rats. Furthermore, the phasic increase normally exhibited by AG-D2 activity in response to acute cold stress was blunted in AGT-and MTP-treated animals. Therefore, we conclude that corticosterone synthesis is necessary in preserving the physiologic response of Ag-D2 activity to cold exposure.

The kinetic characterization of the outer-ring deiodination pathway using rT(3) (rT(3)-ORD) in male, female, and pregnant female livers of an endemic lizard, Sceloporus grammicus, is reported. The ORD pathway does not have the characteristics of deiodinase type II; it is exclusively carried out by deiodinase type I (DI). DI enzymatic activity in lizard liver contains one of the highest activities reported in vertebrates. This activity is sexually dimorphic, with males presenting the highest activity during the reproductive season. The properties of this enzyme correspond to those described in mammals, such as specificity for rT(3), susceptibility to inhibition by 6-n-propyl-2-thiouracil and gold-thioglucose, cofactor requirement, and kinetic pattern. Unlike other vertebrates, the lizard DI exhibits conspicuous stability in the thermal range of 15 to 42 degrees C and in the pH range of 5.0 to 9.0. Male true kinetic constants exhibit a direct correlation with temperature. This is in agreement with short-term adaptation to microenvironmental changes and the feasible expression of enzymatic forms/variants which, together, endow this lizard species with a greater adaptation to natural daily ambient thermal fluctuations.

Mammary deiodinase type I (M-D1) is present only during lactation and exhibits a clear direct correlation with lactation intensity (size of litters). The present work shows that M-D1 is suckling dependent and that intervals between suckling periods no longer than 12 h are essential to maintain this activity. Moreover, we find that with only 15 min of resuckling in 12-h nonsuckled mothers, the 50% decrease in both M-D1 messenger RNA and enzymatic activity could be restored to control values. This restorative effect by suckling may involve pre- and posttranscriptional mechanisms in which norepinephrine and PRL play important roles. Norepinephrine elicits a potent stimulatory effect on M-D1 messenger RNA and enzyme activities, whereas PRL only increases M-D 1 activity and may modulate the enzyme response to norepinephrine. Oxytocin and GH had no effect. These data suggest that the adrenergic nervous system and PRL could directly participate in mammary energetic expenditure, regulating the local T3 supply.

Lactating rat mammary gland expresses a deiodinating activity that, on the basis of kinetic characteristics, corresponds to the socalled 59-deiodinase type I (D1). In the present study we amplified and sequenced several D1 complementary DNA (cDNA) fragments from rat lactating mammary gland. The mammary cDNA was found to be identical to the previously reported rat liver cDNA in the coding region, but 465 nucleotides shorter on its 39-untranslated region, suggesting that the D1 is the same in both tissues. D1 messenger RNA (mRNA) was also detected by reverse transcriptase-PCR in mammary glands from puberal and late pregnant rats, but not in virgin animals. Densitometric analysis showed a close and direct correlation between mRNA content and enzyme specific activity in mammary gland. Our results also show that rat liver contains both D1 mRNA forms and that the large form may respond to the thyroid status. These data suggest a differential and organ-specific expression of these mRNA forms, which could play a role in the functional regulation of D1 activity. (Endocrinology 138: 4248 ‐ 4254, 1997)

Recent molecular cloning studies in mammals and amphibians have demonstrated that the types I, II, and III deiodinases constitute a family of selenoproteins of critical importance in metabolizing T4 to active (i.e. T3) and inactive (i.e. rT3) metabolites. In several tissues of teleost fish, various deiodinase processes have been described, but the structural and functional characteristics of these enzymes and their relationship to the deiodinases present in higher vertebrates remains uncertain. Using a complementary DNA library derived from the liver of the teleost Fundulus heteroclitus, we have identified a complementary DNA that codes for a deiodinase with functional characteristics virtually identical to those of the mammalian and amphibian type II deiodinase. Sequence analysis demonstrates a high degree of homology at both the nucleotide and predicted amino acid levels between the Fundulus clone and these previously characterized type II enzymes, including the presence of an in-frame TGA codon that codes for selenocysteine. These findings demonstrate that the deiodinase family of selenoproteins has been highly conserved during vertebrate evolution and underscores their importance in the regulation of thyroid hormone action.

Deiodinases are major determinants of thyroid hormone tissue availability and disposal. The knowledge of the expression of these enzymes in lower species is important to understand evolutionary and ontogenetic aspects of thyroid hormone action and metabolism. Here we have studied outer ring deiodination in the trout liver using both reverse T3 (rT3) and T4 as substrates. The use of rT3 disclosed two enzymatic components with the characteristics of mammalian types I and II 5′-deiodinases. The high rT3-Km type I 5′-deiodinase activity (180 nm) has a low cofactor requirement (5 mm dithiothreitol) and is relatively sensitive to propylthiouracil inhibition, whereas the low rT3-Km activity was akin to the outer ring deiodination of T4 in these regards. The use of T4 exhibited only a single type of activity with a low Km (0.63 nm), a relatively high cofactor requirement (25 mm dithiothreitol), and propylthiouracil-resistance. Teleosts constitute a unique example of type II activity expression in the liver of an ...

We determined the kinetic parameters as well as the fractional turnover rate (FTr) and half-life (t(1/2)) of rat adrenal gland 5'deiodinase activity (AG-5'D). Adrenal glands from male euthyroid or surgically thyroidectomized (Tx) Wistar rats were homogenized (HEPES, 10MM: ; pH 7.5; sucrose, 0.25M: ; EDTA 1MM: ) and centrifuged at 10,000g for 15 min at 4°C. The resulting crude microsomal supernatants were used for ail measurements of 5'D activity. Using rT(3) (2-500NM: ) the true Km and the Vmax values were of 20.2NM: and 289 fmol of I(-) release/mg protein/h. With T(4) as substrate these values were 5.8NM: and 622 fmol/h/mg protein. Protein inhibitor (cycloheximide 6 mg/100 g wt) administration allowed to determine an FTr of 0.68 h(-1) and a t(1/2) of 1.01 h. Results demonstrate that the greatest 5'D activity in the rat adrenal gland corresponds to isotype II, because the reaction is GTG and PTU-resistant (70-80%), accepts T(4) as a far better substrate than rT(3) (17-fold) and the former thyronine has a 50-90% inhibitory concentration in the 4-100NM: range. Furthermore, rats thyroidectomized for 5 and 15 days showed a conspicuous increase in cerebral cortex and adrenal 5'D-II activity. These characteristics as well as the rapid FTr and short †(1/2) are shared by type II 5'D present in rat pineal, pituitary and brain.

Iodide is a trace element and a key component of thyroid hormones (TH). The availability of this halogen is the rate-limiting step for TH synthesis; therefore, thyroidal iodide uptake and recycling during TH synthesis are of major importance in maintaining an adequate supply. In the rat, the thyroid gland co-expresses a distinctive pair of intrathyroidal deiodinating enzymes: the thyroid iodotyrosine dehalogenase (tDh) and the iodothyronine deiodinase type 1 (ID1). In the present work, we studied the activity of these two dehalogenases in conditions of hypo- and hyperthyroidism as well as during acute and chronic iodide administration in both intact and hypophysectomized (HPX) rats. In order to confirm our observations, we also measured the mRNA levels for both dehalogenases and for the sodium/iodide symporter, the protein responsible for thyroidal iodide uptake. Our results show that triiodothyronine differentially regulates tDh and ID1 enzymatic activities, and that both acute and chronic iodide administration significantly decreases rat tDh and ID1 activities and mRNA levels. Conversely, both enzymatic activities increase when intrathyroidal iodide is pharmacologically depleted in TSH-replaced HPX rats. These results show a regulatory effect by iodide on the intrathyroidal dehalogenating enzymes and suggest that they contribute to the iodide-induced autoregulatory processes involved in the Wolff-Chaikoff effect.

The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have been amply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles. Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepatic D3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of 287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity with chicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is rich in proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves further study. The kinetic characteristics of both native and recombinant PdD3 were similar to those reported for D3 in other vertebrates. True K(m) values for T(3) IRD were 9 and 11 nM for native and recombinant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mM DTT), insensitivity to inhibition by PTU (2 mM), and bisubstrate, sequential-type reaction kinetics. In summary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Furthermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepatic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species in which the dietary T(3) content seems to regulate liver deiodinase expression. Thus, our present results support the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriate overload of exogenous T(3) secondary to feeding in those species that devour the whole prey.

Circulating levels of T4, T3, corticosterone, noradrenaline, and adrenaline, as well as 5'-monodeiodinase activity (5'-MA) were measured in control and hypophysectomized rats acutely exposed to cold environment (15-120 min, 4 C). In addition to the well known activation of the sympathoadrenomedullary system and the hypothalamic-pituitary-adrenal and-thyroid axes, cold exposure was followed by a rapid and sustained increase of 5'-MA in the hypothalamus, and a byphasic course of activation in the adrenal gland in control rats. The adrenal rapid activation (30 min) corresponded to the medulla and the slower activation (120 min) to the cortex. Both, the basal adrenal 5'-MA and the response to cold in adrenal and hypothalamus were 2-fold higher in hypophysectomized rats compared to control. The time course of enzyme activation in these structures suggests that: 1) organ-specific increases in 5'-MA may be associated to a simultaneous rise in their metabolic and/or functional activity, 2) the triggering mechanisms involves an immediate sympathetic signal activating the hypothalamic-adrenal medulla response and a pituitary signal eliciting a slower adrenocortical response, and 3) the compensatory sympathetic hyperactivity after panhypopituitarism contribute to enhance both the adrenal enzyme basal activity and the hypothalamic and adrenal hyperresponse to cold stress.

Until recently, 3,5-diiodothyronine (3,5-T2) has been considered an inactive by-product of triiodothyronine (T3) deiodination. However, studies from several laboratories have shown that 3,5-T2has specific, nongenomic effects on mitochondrial oxidative capacity and respiration rate that are distinct from those due to T3. Nevertheless, little is known about the putative genomic effects of 3,5-T2. We have previously shown that hyperthyroidism induced by supraphysiological doses of 3,5-T2inhibits hepatic iodothyronine deiodinase type 2 (D2) activity and lowers mRNA levels in the killifish in the same manner as T3and T4, suggesting a pretranslational effect of 3,5-T2(Garcia-G C, Jeziorski MC, Valverde-R C, Orozco A. Gen Comp Endocrinol 135: 201–209, 2004). The question remains as to whether 3,5-T2would have effects under conditions similar to those that are physiological for T3. To this end, intact killifish were rendered hypothyroid by administering methimazole. Groups of hypothyroid animals simultaneously received 30 nM of either T3, reverse T3, or 3,5-T2. Under these conditions, we expected that, if it were bioactive, 3,5-T2would mimic T3and thus reverse the compensatory upregulation of D2 and tyroid receptor β1 and downregulation of growth hormone that characterize hypothyroidism. Our results demonstrate that 3,5-T2is indeed bioactive, reversing both hepatic D2 and growth hormone responses during a hypothyroidal state. Furthermore, we observed that 3,5-T2and T3recruit two distinct populations of transcription factors to typical palindromic and DR4 thyroid hormone response elements. Taken together, these results add further evidence to support the notion that 3,5-T2is a bioactive iodothyronine.

The initial characterization of a thyroid iodotyrosine dehalogenase (tDh), which deiodinates mono-iodotyrosine and di-iodotyrosine, was made almost 50 years ago, but little is known about its catalytic and kinetic properties. A distinct group of dehalogenases, the so-called iodothyronine deiodinases (IDs), that specifically remove iodine atoms from iodothyronines were subsequently discovered and have been extensively characterized. Iodothyronine deiodinase type 1 (ID1) is highly expressed in the rat thyroid gland, but the co-expression in this tissue of the two different dehalogenating enzymes has not yet been clearly defined. This work compares and contrasts the kinetic properties of tDh and ID1 in the rat thyroid gland. Differential affinities for substrates, cofactors and inhibitors distinguish the two activities, and a reaction mechanism for tDh is proposed. The results reported here support the view that the rat thyroid gland has a distinctive set of dehalogenases specialized in iodine metabolism.

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.

Type II 5′ deiodinase (5′D-II) activity was assessed in the hypothalamus (HP), pituitary (PIT), and adrenal gland (AG) of adult male rats after two different photoperiodic regimes: (1) cyclic light-dark conditions (LD, 12∶12) or (2) 4 days in continuous darkness (DD). Under cyclic LD conditions HP, PIT and AG 5′D-11 activity showed a significant circadian rhythm (P

The present study analyses the activity of 5′deiodinases type I and II in mammary gland during the breeding and estrous cycle of the rat, and includes indirect evidence that 5′D-I is present only in the alveolar epithelium. Data show that the mammary gland exhibits 5′D-II activity throughout the developmental period and its activity varies along the allometric growth of the gland. 5′D-I is detected during the differentiation stages of the alveolar epithelium (puberty, late pregnancy) and its activity rises significantly (10-fold) 24 h after delivery. Data also show that 5′D-I activity is not present in the fat pads of the gland. These findings suggest that during its differentiation and functional stages, the mammary gland requires an elevated and compartmentalized production of T3.

Serum levels of thyroxine (T4), triiodothyronine (T3) and reverse T3 (rT3) were studied in dry, early (first trimester) and middle (second trimester) lactating purebred Holstein cows. The study encompassed three different seasons: autumn, winter and spring. Under comfortable weather conditions (temperature 22°C; relative humidity, 40%) or moderately hot (28°C; 60%), January and October, respectively, cows in early lactation exhibited significantly lower levels of T4 and T3, and higher values of rT3 than dry or middle lactating animals. In contrast, during May, when environmental temperature increased (34°C; 40%), a clear-cut shift in T3/rT3ratio occurred, and animals in early lactation exhibited the highest T3 and the lowest rT3 concentrations. These findings suggest that in dairy cattle, peripheral thyroid hormone metabolism plays a major role in regulating the homeorhetic responses involved in the maintenance of high priority functions.

The well known metabolic and endocrine adaptive responses accompanying lactation include a change in circulating thyronines that resembles the so-called euthyroid sick syndrome. To analyze the role played by tissue monodeiodination in this state, circulating levels of thyronines as well as hepatic and mammary 5'-monodeiodinative activity (5'MA) were assessed during lactation in rats. Results show that the serum iodothyronine changes that accompany lactation are associated with a significant decrease in hepatic 5'MA and a simultaneous increase in mammary 5'MA. These changes begin within the first postpartum day, are proportional to lactation intensity (litter size), and disappear 48 h after either precocious (1st postpartum day) or natural (21st postpartum day) weaning. These data demonstrate that the compartmentalized change in energy expenditure characteristic of lactation is accompanied by organ-specific and opposite adjustments in hepatic and mammary monodeiodinative pathways.

SummaryProlactin-releasing factor (PRF), activity of rat hypothalamic fragments was increased after incubation in Krebs–Ringer bicarbonate buffer, an effect observed even when puromycin in a concentration sufficient to block new protein synthesis was added. In the presence of a mixture of twenty naturally occurring amino acids, ATP and MG2+, a dialyzed ribosome-free extract of rat hypothalamic tissue showed an increased amount of PRF activity as compared with extracts incubated in the absence of cofactors and amino acids. These observations suggest that PRF is formed by a mechanism not involving new protein synthesis. Since PRF is formed by soluble hypothalamic extracts in the presence of ATP and amino acids, it is proposed that this hypothalamic hypophysiotropic hormone is a polypeptide and is biosynthesized by a “PRF Synthetase.”

The administration of estradiol benzoate, 50 μg, and progesterone, 25 mg, daily for three days has been found to “sensitize” rats to the growth hormone (GH) releasing activity of hypothalamic extracts. At ten minutes following the injection of 90% methanol extracts of 10 porcine hypothalamic fragments, a significant rise in radioimmunoassayable GH was detected (mean increase in pentobarbital anesthetized rats was 28.5 ng/ml ± 12.3 (SE) VS control of 1.6 ng/ml ± 7.9, p < 0.05). The equivalent of 25 hypothalamic fragments gave a significantly larger response. In a series of animals anesthetized with either pentobarbital or ether, 25 or 10-20 hypothalamic equivalents caused significant elevations of RIA-GH and had no depleting effect on pituitary GH concentration. Without steroid pretreatment, significant effects on plasma GH were not observed even with twenty-five hypothalamic equivalents. In steroid pretreated animals, large doses of vasopressin (5 U) and oxytocin (2 U) also induced GH discharge. TRH at a ...

Mammary homogenates from lactating, weaned, pregnant, and nonpregnant rats were analyzed for 5'-monodeiodinase activity (5'-MA). Only lactating glands exhibited significant enzyme activity. Competitive analysis showed that mammary 5'-MA is not inhibited by high (10 microM) concentrations of T4 or T3, but is highly sensitive to prophythiouracil (5 mM). Kinetic parameters demonstrate an acompetitive bisubstrate enzymatic mechanism known as the ping-pong type. The Km and maximum velocity for rT3 were 0.40 microM and 1.42 nmol/mg.min, respectively. These results provide the first evidence that 5'-MA is present in the lactating mammary gland and suggest that this enzymatic activity corresponds to type I enzyme.

Milk yields and the circulating profile of T4, T3 and rT3 were assessed during three different seasons of the year, in first trimester lactating (L) and in dry (D) multiparous holstein cows acclimated to distinct weather conditions. Within the thermoneutral zone (18–28°C; 40–60% RH) and regardless of their geographical location, the thyroid hormone profile in all L-cows (n = 50) resembled the so-called euthyroid sick syndrome (T4, 43.7 ± 7.7 nmol/l; T3, 1.31 ± 0.10 nmol/l and rT3, 0.52 ± 0.08 nmol/l). In both groups of animals the T3/T4 molar ratio was similar within the entire range of climates encompassed in the study. However, both groups exhibited a significant shift in the T3/rt3 molar ratio during cold (10°C; 50%) or hot-dry (34°C; 40%) weather conditions. This shift reaches maximum values (L, 6.5 ± 1.2; D, 7.9 ± 1.0 nmoles/l) under hot-humid conditions (28–42°C; 60–90%). The relative increase of T3 levels from comfortable to cold or hot environmental temperatures, was significantly higher in L than D animals (30 vs 12%, respectively). Furthermore, only L-cows exhibited a significant decrease in the rT3/T4 molar ratio during either type of thermoregulatory demands, as well as a significant increase of T4 values under heat-acclimation. These results suggest that heat-acclimation in dairy cattle does not depress thyroid gland activity, and lend further support to the notion that adaptive thermoregulatory mechanisms in homeothermic vertebrates, involve adjustments in the peripheral monodeiodinative pathways of thyroid hormones.

Plasma prolactin levels in the rat are elevated by reserpine treatment. This effect has generally been attributed to the inhibition of the monoaminergic component of regulation of secretion of prolactin inhibiting factor (PIF). In this study we report that ether stress which produces prolactin release in normal rats was equally effective in stimulating prolactin release in reserpine treated animals whose initial baseline prolactin levels were elevated. These results indicate that the ether stress induced release of prolactin is not due to inhibition of the secretion of PIF, and are compatible with the view that a prolactin releasing substance mediates this acute secretory response.

The biflavanone chamaejasmin has been isolated from the wood of Diphysa robinioides and its structure established by the spectral data of the biflavanone and its hexaacetyl derivative.