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The Sulphur–Iodine (S–I) thermochemical cycle for hydrogen production from water is one of the widest investigated cycles in the world. The S–I cycle is composed of three main sections which correspond to its three main reactions: the Bunsen reaction, the hydriodic acid (HI) decomposition and the sulphuric acid decomposition. The investigation, comprehension and modelling of the complex electrolytic chemistry of the HI solutions is crucial for the process optimization. In particular, the analysis of chemical speciation in the HI solutions is a key factor for the full understanding of the chemical equilibria involved at varying I 2 :HI ratio in the mixtures. The aim of the present study is to use Raman spectroscopy for characterizing the chemical speciation of iodine compounds existing in the mixtures that pertain to the S–I process. For this purpose, Raman spectra of solutions with known HI and I 2 concentration have been recorded. It has been found that the chemistry of these solutions is highly dependent on I 2 :HI molar ratio. Up to 1:1 ratio, I 3 − and its corresponding ion pair HI 3 * are the dominant iodine compounds. At higher values, the formation of higher polyiodine and polyiodides compounds has been suggested.

The synthesis of pyridylmethylene lactams and indolylmethylene-2-indolinones is reported. These compounds were tested for their cytotoxic and positive inotropic activity. The presence of both the effects (coanthracyclinic activity) was confirmed in some of the indolylmethylene-2-indolinones.

In connection with a previous research dealing with the antitumor activity of imidazo[2,1-b]-thiazole guanylhydrazones, this paper reports the synthesis of new derivatives which were tested for antitumor and positive inotropic activity. In most cases the cytotoxic data from the in vitro experiments (HeLa) were in agreement with the antitumor data in vivo (Ehrlich). The active compounds bear a phenyl ring at the 6 position. On the other hand, the most active cardiotonic agents were devoid of the phenyl ring.

Summary This paper describes the synthesis of 6-substituted imidazo[2,1- b ]thiazoles with a lactam ring connected, by means of a methine group, to the 5-position. The pharmacological results show that interesting cardiotonic activity is obtained when the lactam ring is pseudothiohydantoin ( 8 ) or barbituric acid ( 9 ). Even the substituent at position 6 plays an important role in the pharmacological behavior of these derivatives. The following activity rank order was observed: phenyl > methyl > chlorine.

L’elettroforesi della tireoglobulina 19 S bovina in condizioni denaturanti, non riducenti ha rivelato, oltre ai monomeri di tireoglobulina 12 S, quattro specie distinte di dimeri covalenti. Dimeri diversi esposti all’azionedel 2-mer-captoetanolo hanno mostrato composizione in polipeptidi simile, ma diversa suscettibilita alla dissociazione per mezzo della riduzione dei ponti disolfuro intermonomerici. L’abbondanza di questi dimeri era correlata al contenuto in iodio della tireoglobulina. I vari tipi di dimeri covalenti della tireoglobulina differiscono, apparentemente, per il numero e la posizione dei ponti disolfuro intermonomerici.

The syntheses of 4 different fused imidazoles bearing a 2,5-dimethoxyphenyl group or a pyridyl group are reported. All the heterocycles, except 2a , display positive inotropic activity. Some are approximately equipotent with sulmazole, and the imidazo[2,1- b ]benzothiazole that bears a 4-pyridyl group is the most active of whole series.

The syntheses of 4 different fused imidazoles bearing a 2,5-dimethoxyphenyl group or a pyridyl group are reported. All the heterocycles, except 2a , display positive inotropic activity. Some are approximately equipotent with sulmazole, and the imidazo[2,1- b ]benzothiazole that bears a 4-pyridyl group is the most active of whole series.

Microcystin diversity in a Planktothrix rubescens population from Lake Albano (Central Italy). Toxicon 48, 160-174. Elsevier. Abstract The cyanobacterium Planktothrix rubescens Anagnostidis & Komarek (previously Oscillatoria rubescens DC ex Gomont) is present in several Italian lakes and it is known to produce cyanotoxins. The dynamics and toxin production of P. rubescens population in Lake Albano, a volcanic crater lake in Central Italy, has been studied for 5 years (January 2001–April 2005). Winter–spring super.cial blooms with frequent scums were observed every year. Total microcystin (MC) levels were measured from April 2004 to October 2005 by liquid chromatography-tandem mass spectrometry. MC levels up to 14.2 mg/l were measured, with high concentrations found in summer at a 20–25m depth. The intracellular toxin content varied between 1.5 (surface, January 2004) and 0.21 pg/cell (surface, May 2004). Six different MCs were detected, the most abundant being two desmethyl-MC-RR isomers. Of the 13 water wells monitored in the Lake Albano area, two of them showed MC contamination during winter, con.rming the ability of these toxins to migrate through groundwater towards public water sources. These results highlight the need for further studies on the mobility and fate of these pervasive cyanobacterial toxins.

Melaleuca alternifolia Cheel essential oil (TTO) and its major component terpinen-4-ol were examined against a large number of clinical isolates of Staphylococcus aureus to establish their anti-staphylococcal activities. Classic and established procedures were used to study M.I.C., time-kill curves, synergism and mutational frequency. The anti-staphylococcal activity of terpinen-4-ol and TTO were superior to those of antibiotics belonging to the major families (all the tested drugs are for topical use or included in ointments, eye drops or used during surgery); terpinen 4-ol and TTO were active against strains resistant to mupirocin, fusidic acid, vancomycin, methicillin and linezolid. TTO and terpinen-4-ol were bactericidal as revealed by time-kill curves; the frequency of mutational frequency to TTO was < 2.9×109. The study demonstrates good anti-staphylococcal activity of TTO and terpinen-4-ol against a large number of S.aureus isolates and suggests the possible application of these agents for topical treatment of staphylococcal infections. This is the first extensive study on the anti-staphylococcal activity of TTO. The results suggest that this compound may have application as a topical agent for the control of superficial staphylococcal infections, including activity against organisms resistant to antibiotics which can be used, or are specific, for topical use.

In an investigation of the in vivo clastogenic potential of the food colouring erythrosine (ER), male B6C3F1 mice were treated by ip injection at doses of 50, 100 and 200 mg/kg, repeated 24 hr apart. Signs of toxicity were observed at the highest dose of ER administered. The three cytogenetic endpoints analysed were sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs), micronuclei in bone marrow polychromatic erythrocytes (PCEs), and micronuclei in peripheral blood reticulocytes (PBRs). SCE frequencies in PBLs were 4.13, 4.58, 4.33 and 4.60 SCE/cell at 0, 50, 100 and 200 mg ER/kg, respectively. At the same doses, the frequencies of micronucleated PCEs were 3.5, 3.2, 2.0 and 2.5/1000 PCEs. Micronuclei in PBRs ranged from 1.2 to 3.6 and from 1.4 to 3.0/1000 PBRs in control and treated mice, respectively. These results indicate that ER is inactive as a clastogen in mouse blood and marrow cells. This result supports the hypothesis of a non-genotoxic mechanism for ER carcinogenicity.

Iodine incorporation into thyroglobulin is known to occur within the lumen of the thyroid follicle. Since incorporation of sialic acid, which occupies a terminal position in the oligosaccharide chains, is also a later event in thyroglobulin synthesis, the possibility that sialic acid might be incorporated after thyroglobulin secretion was investigated. In one experimental approach normal rat thyroid hemilobes were incubated with radioactive precursors. Thyroglobulin, analyzed by equilibrium centrifugation in RbCl, had a median density which varied according to the moiety labeled in the following increasing order: leucine smaller than galactose smaller than sialic acid smaller than iodine. The molecules having the highest density were labeled only with iodine. In the second approach, thyroid hemilobes were taken from rats treated with cycloheximide for 16 hours to stop protein synthesis and allow nascent molecules to be secreted, and incorporation of precursors into thyroglobulin was analyzed by sucrose gradient centrifugation. Leucine incorporation was 6% of control but the amino acid was found in the NH2-terminal position. N-Acetylmannosamine (sialic acid precursor) and galactose incorporation were also completely inhibited whereas iodine incorporation was 10% of control. Incorporation was not restored by thyrotropin treatment, and the sialyltransferase and iodination systems were reduced only to 50 to 70% of control. The results indicate that sialic acid is incorporated only in nascent thyroglobulin and not in thyroglobulin molecules already secreted into the follicular lumen. A large fraction of the iodine incorporation also seems to occur in newly synthesized thyroglobulin.

This paper explores the irritancy potential of n-alkyl benzoate preservatives (methyl benzoate. MB; ethyl benzoate, EB; n-propyl benzoate, n-PB; n-butyl benzoate, n-BB), allowable in cosmetic formulations, Comparative experiments were carried out involving repeated daily application for all com-pounds (up to maximum of 6 days) on clipped rabbit skin in 2 distinct regions: the dorsum (0.5 ml per treatment) and the external surface- of the- outer ear (0.2 ml per treatment). According to the different anatomo-physiological features, benzoates elicited dissimilar reactions in the dorsum and the external ear. In the first mentioned area, there were widespread inflammatory reactions accompanied, in some cases, by necrotic changes and slight epidermal hyperplasia; on the other hand, hyperplasia was the most prominent and earliest alteration in the car. The irritancy potential was closely related in the duration of treatment as well to the increase in the molecular weight of benzoates.

The iodoproteins from goitrous rat thyroid glands (300–800 mg), obtained by prolonged (20 months) and severe iodine deficiency, were isolated and characterized. The animals were given a low iodine diet (2 μg 1/100 g diet) containing 125I for 81 days (approach to equilibrium) and then they were pulse—labeled with 131I and 3H—leucine 2 hr prior to sacrifice. Thyroglobulinlike iodoproteins represented only 5% of the total protein in the crude soluble extract with 19 S comprising 90% and 27 S 10%. There was almost no iodine in 19 S while 27 S contained 0.12% iodine. Therefore a large proportion of the iodine stored in the soluble iodoproteins was due to the iodine in the 27 S protein. The turnover of iodine in 27 S, however, was much slower than in 19 S. In these severely iodine deficient animals the distribution of soluble thyroid iodoproteins was not significantly different from that observed in normal glands; the low iodine content, however, affects the stability of thyroglobulin. The iodinefree 19 S was m...

Iodine-labeled 4-hydroxy-3,5-diiodophenylpyruvic acid was prepared and converted into its hydroperoxide (thyroxine precursor). Thyroglobulin was permitted to react with the hydroperoxide and the nature and amount of the iodothyronine residues formed in this model reaction for the biosynthesis of thyroxine were investigated. Iodine-rich bovine thyroglobulin (54 atoms I per molecule) and iodine-poor human thyroglobulin (6–10 atoms I per molecule) were used. After the coupling, the thyroglobulin was analyzed by chromatographic fractionation of its pronase digest. The analysis showed the formation of about 1 and 2 iodothyronine residues per molecule in the coupling reaction with iodine-poor and iodine-rich thyroglobulin, respectively. The hydroperoxide reacts with protein-bound diiodotyrosine and monoiodotyrosine. Hence both thyroxine and 3,3′,5′-triiodothyronine residues are formed. The molar ratio of newly formed thyroxine to 3,3′,5′-triiodothyronine residues was 4 in iodine-rich thyroglobulin (diiodotyrosine to monoiodotyrosine molar ratio of 0.9) and 0.4 in iodine-poor thyroglobulin (diiodotyrosine to monoiodotyrosine molar ratio of 0.2). Thus, the hydroperoxide reacts more efficiently with diiodotyrosine than with monoiodotyrosine residues. The amount of iodothyronine residues formed by coupling with the hydroperoxide (intermolecular reaction) was compared with that formed when thyroglobulin is iodinated to a comparable iodine level (intramolecular reaction). At high iodine levels both model reactions proceed with similar efficiencies, but at low iodine levels coupling with the hydroperoxide of 4-hydroxy-3,5-diiodophenylpyruvic acid is considerably more efficient.

In the present review the problem of the evolutionary level at which the biosynthesis of thyroid hormones first appeared in the zoological scale is discussed. Thyroid hormones are certainly present in all Chordata, Urochordata, Cephalocordata, and Vertebrata. However, although concentration and organification of iodine are widespread processes occurring throughout the animal kingdom, the synthesis of thyroid hormones, i.e., iodothyronines, has not been clearly established below the Chordata. The biochemical mechanism leading to hormone synthesis follows the same general pattern from its first appearance in the Protochordata. The coupling of iodotyrosines to yield iodothyronines occurs within the polypeptide chains of specific proteins. In vertebrates, from the cyclostomes up to the mammals, the polymerization of similar, but perhaps not identical, subunits, leads to the formation of a family of thyroid proteins of increasing molecular size, i.e., 12 S, 19 S (thyroglobulin), and 27 S iodoproteins.

Beef or hog thyroglobulin was iodinated with varying amounts of 125ICl. The iodinated thyroglobulin was then permitted to react with 4-hydroxy-3,5-diiodophenylpyruvic acid in the presence of O2. Radiochromatographic analysis of an enzymic hydrolysate of the reaction mixture showed that labeled thyroxine and 3,3′,5′-triiodothyronine residues had been formed in the course of the reaction, apparently by the coupling of labeled diiodotyrosine and monoiodotyrosine residues with 4-hydroxy-3,5-diiodophenylpyruvic acid.

A tunicate, Ciona intestinalis L., binds very actively I/sup 131/-ions. The labeled iodine is preferentially located (75%) in the external layer of the tunic. The iodine metabolism is there chiefly oriented toward the biosynthesis of iodothyronines (thyroid hormones). The presence of free thyroxine and 3,5,3'- triiodothyronine in the tissues of Ciona has been established. Biogenesis of 3,5,3'-triiodothyronine and thyroxine proceeds chiefly in the tunic, in which three fractions of iodoproteins were characterized. Two of these, one soluble in various media, the other bound to insoluble structure, contain 3,5,3'- triiodothyronine, thyroxine and their precursors, 3-monoiodo- and 3,5- diiodotyrosine. The four iodinated amino acids are freed from these proteins by action of pancreatic proteinases and papain. An insoluble residue of iodinated scleroprotein, not hydrolyzed by proteinases, does not take part to endocrine activity; 3-monoiodotyrosine and 3,5-diiodotyrosine only are found in the products of its alkaline hydrolysis. The mechanism of hormonogenesis seems identical in all chordata (urochordata, cephalochordata, and vertebrates). As thyroid hormones are found in these and not in invertebrates lower than urochordata, their appearance in this subphylum can be considered as a fundamental step of biochemical evolution of the thyroid function. Biosynthesis of iodothyronines is characteristic of chordata. It proceeds inmore » all of these, including protochordata, by the same mechanism, but under conditions submitted to the evolution of the secretory tissue through a series of steps, the last of which is the thyroid follicle of vertebrates. (auth)« less

Resume 1. (1) Les larves de Petromyzon planeri Bl. (Ammocoetes) fixent les iodures marques par 131I ajoutes a l'eau dans laquelle ils sont immerges. Ce processus, assez intense pendant 4 jours a +18°C, devient par la suite beaucoup moins actif. 131I est concentre preferentiellement dans la region de l'endostyle et dans la chorde dorsale. L'endostyle renferme une ebauche thyroidienne fonctionnelle dont l'activite a eteetudiee par radiochromatographie et radioelectrophorese des constituants iodes marques de la region endostylaire et des tissus recepteurs des hormones. 2. (2) La region endostylaire renferme de la thyroxine, de la 3 : 5 : 3′-triiodothyronine et leurs precurseurs, la 3 : 5-diiodotyrosine et la 3-monoiodotyrosine, incorporees dans une proteine, d'ou une hydrolyse proteinasique. On ne trouve, par contre, aucune iodoproteine dans les tissus recepteurs (ensemble des tissus mous en dehors de l'endostyle), dont la thyroxine et de petites quantites de 3 : 5 : 3′-triiodothyronine peuvent etre extraites san hydrolyse proteinasique. 3. (3) Les rapports quantitatifs entre les acides amines marques de la region endostylaire et des tissus recepteurs sont tres variables suivant le temps d'incubation avec 131INA. L'hormonogenese, bien que plus lente, releve d'un meme mecanisme dans l'endostyle des larves du Cyclostome etudie et dans le corps thyroide des Mammiferes. 4. (4) Le transport sanguin des hormones parait etre assure par une proteine plasmatique capable de fixer la thyroxine.

To elucidate the mechanism of induction of bacterial spore germination by l -alanine and by hydrogen peroxide, the effect of germination of some of their metabolites has been studied. The degree of germination has been evaluated on the basis of the loss of heat resistance of the germinated spores. Pyruvate or pyruvate associated with glutamate do not induce germination, while pyruvate associated with ATP caused a noticeable degree of germination (25%) which does not depend on the concentration of the two substances. Neither adenosine triphosphate nor phosphopyruvate alone promote germination. The germination induced by l -alanine is inhibited by p -chloromercuribenzoate and by Atebrin, while it is not influenced by cyanide or by the hydrazide of isonicotinic acid. The germination induced by hydrogen peroxide is inhibited by cyanide and not by p -chloromercuribenzoate or Atebrin. In the cell-free extracts of the vegetative forms of B. subtilis , a DPN-linked l -alanine dehydrogenase activity is detectable, which is completely inhibited by p -chloromercuri benzoate at a concentration of 1.1 · 10 −4 M . Such activity is absent in the cell-free extracts of dormant spores and of spores germinated by l -alanine. On the basis of the results, it is concluded that the germination of bacterial spores seems to be dependent on the utilization of the active substances in metabolic reactions essential to the cells and on the activation of the respiratory system and of the energetic metabolism of the cell.

A native thyroid protein from guinea pig with a sedimentation coefficient of 12 S has been purified by sucrose gradient centrifugation. Its iodine content is one-tenth that found in thyroglobulin (19 S) obtained from the same preparation. It has been shown that this naturally occurring thyroid protein is not derived from and does not dimerize to 19 S thyroglobulin. While it does not interchange with undissociated 19 S, it combines with the 12 S subunits derived from 19 S to form hybrid molecules of thyroglobulin. It is inferred from the hybridization experiments that thyroglobulin consists of two different 12 S subunits, one of them being equivalent to the naturally occurring 12 S molecule.

Thyroglobulin, the major protein component of normal thyroid glands, contains more than 80% of the thyroidal iodine and has been considered the only source of thyroid hormones (1). Aqueous thyroid extracts contain, besides thyroglobulin, other proteins in variable amounts. Essentially, four methods have been used for the characterization of the various components in such extracts and for the purification of thyroglobulin: salting out, electrophoresis, column chromatography, and ultracentrifugation. Salting out with ammonium sulfate or potassium phosphate (2) is the classical method for preparing thyroglobulin. However, salting out preparations are always contaminated with compounds that sediment faster than thyroglobulin, the 19 S ultracentrifugal component of thyroid extracts (3-5). Electrophoretic methods likewise yield preparations of thyroglobulin known to be heterogeneous when analyzed in the analytical ultracentrifuge (4, 6-9). Column chromatography on DEAEcellulose gives highly purified thyroglobulin (4, 10) but the yield is rather poor. The main disadvant.age of this method is that thyroglobulin itself is fractionated on these columns according to its iodine content. The ultracentrifugally homogeneous fraction obtained by this method has a considerably lower iodine content than the whole thyroglobulin (4, 11-13). Ultracentrifugation is one of the most useful analytical procedures, but preparative moving boundary ultracentrifugation (3) requires multiple runs to obtain good yields of highly purified thyroglobulin. All the methods now available for the purification of thyroglobulin and for the analysis of crude or partially purified thyroid estracts therefore present major shortcomings. The need for suitable methods for the analysis of thyroid extracts, as well as for the preparation of highly purified thyroglobulin, led to the work to be reported here. Methods of fractionation based essentially on molecular size were found to be most useful. Filtration through granulated agar or dextran gel columns was used for the purification of thyroglobulin; ultracentrifugation in a linear density gradient was employed for both analytical and preparative purposes.

Interest concerning the possible adverse effects of cosmetic products is increasing and at the same time toxicologists are faced with the problems arising from in vivo testing and the growing development of alternative methods. A series of n -alkyl benzoates has been tested on a continuous cell line (HEp-2). Dose-effect correlations have been established for methyl, ethyl, n -propyl and n -butyl benzoates by determining the total protein content of the cells at 24 hr. The toxicity ranking of these benzoates according to the IC 50 has been shown to be butyl > propyl > ethyl > methyl benzoate. Experiments carried out on time-effect correlation and on colony-forming ability seem to suggest that the damage does not increase with time and is probably caused mostly by an initial selective effect on the cell population.

Since 1978 this department has been engaged in carrying out investigations on the acute and subchronic toxicity of 3,5-dinitro- 4-chloro-α,α,α-trifluorotoluene (DNCTT) and 3-nitro-4-chloro- α,α,α-trifluorotoluene (NCTT) which in 1977 were detected as surface water contaminants in northern Italy. This paper is a progress report of an on-going study of the subchronic toxicity of DNCTT. Diets containing different levels of DNCTT (20; 200; 2,000 ppm) were administered to different groups of young male and female rats for 28 days. No animal deaths were observed at any dose levels. A slight reduction of body weight increase, and of food and water intake was observed in both male and female rats, particularly at the highest dose level tested (Fig. 1). A dose-related increase was observed in liver and spleen relative weights in both sexes, but it was significant (p < 0.005) only at the highest dose level (liver: m 6.6%, f 22.3%; spleen: m 41%, f 27%). Significant alterations were also seen in blood parameters. Glycemia was increased dose-dependently in both sexes (2,000 ppm: m 43%, f 66%) as were triglycerides in the treated females. Cholesterol and triglycerides were significantly higher (40.7% and 31.3% respectively) only in male animals treated with 2,000 ppm of DNCTT. Animals treated with this dose level also showed a significant decrease in haemoglobin (m 9.3%, f 12%) and erythrocyte count (m 5%, f 18%). Cage-side observations, carried out on a daily basis, did not show any major change in the treated animals with the exception of the finding a yellow coloration of urine and fur seen only at 2,000 ppm of DNCTT. The available findings on the toxicity of DNCTT are closely related to those observed in a previous similar investigation on the oral toxicity of NCTT and are consistant with a possible role of DNCTT as an inhibitor of the mitochondrial respiratory chain.

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.

The use of a sample cleanup and extraction device and a column switching technique allowed us to propose an automatized method for the determination of plasma xanthines1.

1. 1. The movements of I− and other anions in Ehrlich ascites tumor cells were studied as a model to explain the restricted anion space of certain cells. 2. 2. At 38° all the anions tested (I−, Br−, ReO4−, WO42−) penetrate only one-quarter of the cell volume. For I− this value, constant up to 6 h, is independent of the anion concentration in the medium from less than 1 μM to 0.124 M and is insensitive to anions that inhibit thyroidal I− transport competitively. 3. 3. At 4° the restriction of the I− (and Br−) compartments (but not that of ReO4− or WO42−) is abolished and the I− space approaches the water content of the cell. Upon exposure of the cold-loaded cells to 38°, a net efflux of the halide is observed until the steady-state value of one-quarter of the cell volume is again attained. 4. 4. The temperature-dependent efflux of I− is sensitive to various metabolic inhibitors, but, in ascites fluid, at a rather high concentration. 5. 5. Cardiac glycosides and quinidine markedly decrease the ability of ascites cells to restrict the anion space. The same effect is produced by nucleoside triphosphates, particularly ATP, ITP and UTP. 6. 6. The behavior of I− and Na+ was compared under identical conditions. Although certain similarities exist, a number of differences were noted. 7. 7. The evidence presented is consistent with the hypothesis that the halide restriction in ascites cells is due to a metabolism-dependent efflux.

Hog and bovine 27 S thyroid iodoprotein, incubated for prolonged periods at 4 °C and neutral pH, formed 19 S and 2.5 S species ( M r 31,000) which were isolated by gel filtration. Native 19 S thyroglobulin, the major thyroid iodoprotein, remained unchanged under the same conditions. The iodine content of the 2.5 S species was 1.5 times that of 27 S and there were significant differences in amino acid composition between 2.5 and 27 S. Hog 27 S (after separation of 2.5 S) dissociated into two species in 6 m guanidine-HCl. They were isolated by sucrose density-gradient centrifugation and found to have sedimentation coefficients of 2.6 S ( M r 44,000) and 4.9 S ( M r 65,000–85,000). The appearance of approximately 10 amino terminal groups on analysis of 27 S suggests that peptide bonds were cleaved during incubation since no amino terminal groups were found in freshly prepared 19 or 27 S. Freshly prepared hog 27 S was found to be more sensitive to tryptic digestion than 19 S. Since only a limited number of discrete species were formed during the incubation of 27 S, there appear to be preferential sites of cleavage, suggesting the possible existence of intrachain, repeating, homologous sequences in the 27 S iodoprotein.

Publisher Summary This chapter discusses the chemistry and the biosynthesis of thyroid iodoproteins. The thyroid is almost unique among other endocrine glands in that it possesses, within its basic morphological structure, the follicle, rather large stores of hormone in a form readily available to the organism. The thyroid iodoproteins represent the form in which thyroid hormones are stored in the gland; the availability of these hormonal deposits is dependent upon the hydrolysis of the iodoproteins and the subsequent secretion into the blood-stream of the biologically active iodoamino acids, thyroxine, and 3,3',5-triiodothyronine. However, the thyroid iodoproteins represent not only the form in which the hormones are stored but also the molecular site of hormonal biosynthesis. Small fragments of thyroglobulin have been obtained by proteolytic digestion but no peptides have been sequenced. When bovine thyroglobulin was hydrolyzed by trypsin, chymotrypsin, papain, subtilisin, or pepsin, no intermediate sized molecules were observed by ultracentrifugation.

The time course of iodination of individual thyroid iodoproteins has been investigated by “equilibrium” labeling in rats and “pulse” labeling in rats and man. Although the “equilibrium” experiments demonstrated little difference between the rate of incorporation of iodine into these poteins after two days, pulse label experiments at earlier times indicated that thyroglubulin (19 S) was labeled at a faster rate than the 27-S iodoprotein. The 27-S protein, which contains more iodine, becomes more highly labeled than thyroglobulin during “equilibrium” labeling. Radiochromatography after hydrolysis revealed no significant difference in the relative distribution of iodoamino acids in the 19-S and 27-S proteins at varied times after labeling. Ultracentrifugal heterogeneity of thyroglobulin has been demonstrated by density-gradient centrifugation. A slower-sedimenting fraction containing less iodine was separated from a more highly iodinated, faster-sedimenting one. A correlation seemed also to exist between the chromatographic behavior of thyroglobulin on DEAE-cellulose and the sedimentation properties of thyroglobulin. The faster- sedimenting molecules were more strongly retained on DEAE-cellulose, and labeling in vivo showed a positive correlation between the time of iodination and the sedimentation rate. It is concluded that the faster-sedimenting thyroglobulin molecules are more dense, contain more iodine, are more negatively charged, and are iodinated later than the slower-sedimenting ones.

The effect of in vitro ultraviolet irradiation at 2537 A on the structure of highly purified human thyroglobulin has been investigated by following the ultracentrifugal pattern and the appearance of free sulfhydryl groups in the irradiated protein. Thyroglobulin (19 S) dissociates into two 12 S subunits when it is irradiated with doses of a few hundred Einsteins per mole. The quantum yields of dissociation vary between 0.5 × 10−3 and 2 × 10−3, depending on the experimental conditions. At higher ultraviolet doses, the protein is partially aggregated and denatured. The extent of dissociation is largely dependent on the ionic strength and the pH of the medium. Doses up to 1,500 Einsteins per mole cause only a partial (30%) splitting of the 19 S molecules, at neutral pH in 0.1 M KCl, whereas at pH 10 and low salt (Γ/2=0.0042), the same dose produces almost complete dissociation into 12 S subunits. Ultraviolet light also causes the rupture of some of the disulfide bonds of native thyroglobulin. On irradiation with several hundred Einsteins per mole protein, as many as 21 –SH groups per molecule can be titrated. The quantum yields for –SH production and destruction of cystine residues are both 0.05. No direct correlation has been found between the extent of dissociation of thyroglobulin and the number of –S–S– bonds cleaved, nor between the reassociation of the 12 S subunits and the reoxidation of –SH groups. It is concluded that two 12 S units present in human thyroglobulin are not linked by interchain disulfide bridges; the disulfides cleaved by ultraviolet irradiation, however, may play an important role in maintaining the subunit organization of the protein molecule.

In several neuroblastoma cell lines, retinoic acid (RA)–induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell–derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2′-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell–derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies. (Mol Cancer Res 2006;4(7):481–8)

We have used the polyoma middle T oncogene to immortalize cells from rat embryo encephalon. Immunostaining experiments with monoclonal antibodies demonstrated that the cells of one of the obtained lines, named CEINGE CL3, are stained by anti-vimentin and anti-S100 antibodies, are not stained by anti-neurofilaments (NF) or anti-glial fibrillary acidic protein (GFAP) antibodies. Only a subset of the CEINGE CL3 cells (20–30%) is stained by an anti-galactocerebroside antibody. Northern blot analysis demonstrated that these cells express low levels of proteolipid protein mRNA, whereas polymerase chain reaction (PCR) amplification failled to evidentiate the presence of both NF and GFAP mRNAs. Either retinoic acid or forskolin treatments or a combination of them are able to induce morphological changes that are accompanied by a complete growth arrest.