Specificity of submaxillary gland sialyltransferases

A method has been developed to determine the activities of specific sialyltransferases by analysis of the products of the reaction. This method, which utilizes high performance liquid chromatography, distinguishes addition of sialic acid to the N-acetylgalactosamine vs. galactose residues of the mucin disaccharide Galβ(1→3)GalNac, and can be used to distinguish formation of the 3′- and 6′-isomers of sialyllactose. For the bovine, ovine, and porcine submaxillary extracts, more than 95% of the activity with asialo ovine submaxillary mucin is due to formation of NeuAc α(2→6)GalNAc. With lactose as the acceptor, more than 95% of the α(2→3) isomer is produced. Activity with asialofetuin is due solely to the O-linked chain, with relative activity toward the galactose vs. GalNAc residues of 0.32, 1.5, and 0.10 for bovine, ovine, and porcine, respectively. The rat submaxillary gland extract showed equal formation of 3′- and 6′-sialyllactose, and very low activity with asialo ovine submaxillary mucin. However, at least 40% of the activity toward the Galβ(1→3)GalNAc disaccharide of asialofetuin was directed toward the GalNAc residue. The relative preference of the N-acetylgalactosaminide α(2→6) sialyltransferase for a monosaccharide vs. a substituted GalNAc may play a role in regulation of chain length during mucin synthesis.     

Iodide transport in isolated cells of mouse submaxillary gland

A method has been developed to isolate cells from the submaxillary gland of mouse by treatment with pronase. Three fractions of cells have been isolated having almost equal iodide concentrating activity. The isolated cells show time dependent uphill transport of iodide. The transport is substrate-saturable, having aK _m value of 0.3 μM for iodide. The transport is sensitive to antithyroid drugs, metabolic inhibitors and to some extent to ouabain. Pseudohalide such as thiocyanate competes with the transport of iodide. Thyroid hormones or thyroid stimulating hormone have no significant effect on the iodide transport in these cells.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

Microwave-induced increase of water and conductivity in submaxillary salivary gland of rats

Hypersalivation is an important mechanism for heat dissipation by animals without sweat glands. The water content and conductivity (at 20 kHz) in sub-maxillary salivary gland (SSG) and in other tissues were investigated in adult male rats exposed to microwaves (2880 MHz, 1.5 μs pulses at 1000 Hz) or to conventional heat at 40 °C. Eighty rats in one series were exposed, one at a time, for 30 min to microwaves producing a specific absorption rate (SAR) of 4.2,6.3,6.8,8.4,10.8 or 12.6 W/kg. Fifty rats were sham-exposed under similar environmental conditions. In the second series, ten rats were sham-exposed, 33 rats were exposed, one at time, for 15, 30 or 60 min to microwaves at a SAR of 9.5 W/kg, and 32 rats were exposed for similar periods to conventional heat at 40 °C. In rats of the first series colonic temperatures were elevated significantly at a SAR of 4.2 W/kg, while SSG water content and conductivity increased significantly at SAR values of 6.3 W/kg and higher. In the second series of experiments increases in colonic temperature and SSG water content were greater after 15 and 30 min of microwave exposure than after exposure to heat. Also, SSG conductivity was significantly depressed by heat and significantly increased by microwaves after exposure for 15 or 30 min. The results support the hypothesis that water content and conductivity of SSG of rats can be used as a sensitive specific test of a microwave induced thermal response.     

In vitro effects of thyroid hormones on ectonucleotidase activities in synaptosomes from hippocampus of rats

1. Studies have shown that adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. Since the ectonucleotidase pathway is an important source of adenosine extracellular, in the present study the in vitro action of T3 and T4 hormones on ectonucleotidase activities in hippocampal synaptosomes was evaluated.2. T3 (Triiodo-l-thyronine) significantly inhibited, in an uncompetitive manner, the ATP and ADP hydrolysis promoted by ATP diphosphohydrolase activity in hippocampal synaptosomes of adult rats.3. In contrast, T4 (Thyroxine) only inhibited ATP hydrolysis in an uncompetitive mechanism, at the concentrations tested (100–500 M), but at the same time did not affect ADP hydrolysis.4. In the present study, we also investigate the in vitro effect of T3 and T4 on 5-nucleotidase activity. However, there are no changes in the activity of this enzyme in the presence of T3 and T4 in the hippocampal synaptosomes of rats.5. These results suggest that thyroid hormones could be involved in the regulation of ectonucleotidase activities, such as ecto-ATP diphosphohydrolase and ecto-ATPase, possibly exerting a modulatory role in extracellular adenosine levels.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.     

99TcmO4 as an indicator for specialized cell function in organ cultures of the human submaxillary gland

Summary Salivary gland tissue was cultured in vitro and viable cells were present during 14 days. The specific glandular cell function was estimated from the capacity to accumulate ⁹⁹Tc^mO₄ from the culture medium. The morphology of the cultured specimens was examined by light and electron microscopy. Structural changes and loss of the ⁹⁹Tc^mO₄ accumulation capacity increased with increasing culture time in vitro. A correlation was observed between the loss of ⁹⁹Tc^mO₄ accumulation capacity and the disappearance of zymogen granules from the cultured cells. However, the causative link between these two phenomena requires further analysis.Supported by grants from Karolinska Institutet     

Induction of metamorphosis in the sand dollar Peronella japonica by thyroid hormones

The larva of the sand dollar Peronella japonica lacks a mouth and gut, and undergoes metamorphosis into a juvenile sand dollar without feeding. In the present study, it was found that thyroid hormones accelerate the metamorphosis of P. japonica larvae. The contents of thyroid hormones in larvae increased gradually during development. Thiourea and potassium perchlorate, inhibitors of thyroid hormone synthesis, delayed larval metamorphosis and simultaneously repressed an increase in the content of thyroxine in the larval body. These results suggest that the P. japonica larva has a system for synthesis of thyroid hormones that act as factors for inducing metamorphosis.     

In vitro pregnenolone metabolism by mouse adrenal gland: II-Biosynthesis of androgens

Adrenal gland homogenates from four different strains of mice were incubated with (4-14 C)-pregnenolone and a NADPH generating system. The most important androgen synthesized was dehydroepiandrosterone; testosterone and progesterone were synthesized to a lesser extent and the production of androstenedione was very low. The highest synthetic activities were found in the high mammary tumor strain of mice (C3H x RIII) Fl; they were increased by ovariectomy, particularly when performed at two months of age. In the other strains, they were lower, specially in the low mammary tumor strain C 57 BL. However, the 3 beta-hydroxysteroid dehydrogenase / delta 5, 4 isomerase activity was not modified by ovariectomy in the high mammary tumor strain whereas it was increased in the low mammary tumor strains. These results indicate that the androgen synthesis in mouse adrenal depends on factors such as age, sex, endocrine status (ovariectomy) but also on susceptibility to mammary tumor development.     

Cytochemical localization of hydrogen peroxide generating sites in the rat thyroid gland

Sites of H₂O₂ generation in lightly prefixed, intact thyroid follicles were studied by two cytochemical reactions: peroxidase-dependent DAB oxidation and cerium precipitation. In both cases reaction product accumulated on the apical surface of the follicle cell at the membrane-colloid interface. The former reaction was inhibited by the peroxidase inhibitor, aminotriazole; both reactions were blocked by the presence of catalase. NADH in the medium slightly increased the amount of cerium precipitation. The ferricyanide technique for oxidoreductase activity was also applied; reaction product again was associated with the apical surface. These results strongly imply that the follicle cells have a NADH oxidizing system generating H₂O₂ at the apical plasma membrane.     

Nutritional control of thyroid morphogenesis through gastrointestinal hormones

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Immunological characterization of soluble peroxidases from rat tissues including preputial gland

A highly active soluble peroxidase has been identified in the preputial gland of rats and characterized immunologically along with other soluble peroxidases of a number of rat tissues such as submaxillary gland, exorbital lacrimal gland and also of the uterine fluid of the estrogen treated rats. All these peroxidases have the native molecular weight around 73K as determined by gel filtration on Sephadex G-150. An antiserum raised against the pure bovine lactoperoxidase interacts with all these soluble peroxidases and immunoprecipitates the enzyme activity in a similar fashion when titrated against varied concentration of the antiserum. Following electrophoretic transfer to nitrocellulose by Western blotting, the antiserum crossreacts with the preputial, submaxillary and lacrimal gland protein of molecular weight around 73K and with the uterine fluid protein of molecular weight of 80K. An additional crossreacting protein of molecular weight of 80K is also evident in the lacrimal gland. All these enzyme preparations, however, contain another immunoreactive protein of molecular weight of about 64K. While 73–80K molecular weight interacting proteins may represent different forms of peroxidase, presumably with varied carbohydrate moieties, 64K molecular weight protein may be a precursor of the peroxidase which after posttranslational modification such as heme conjugation and glycosylation leads to formation of native enzyme. Rat harderian gland, unlike bovine origin, does not contain any detectable peroxidase activity. The immunoblot does not show the presence of any immunoreactive protein around 73K except the 64K molecular weight protein indicating that this gland can not synthesize the native peroxidase from this precursor probably due to some block in posttranslational modification.     

Genetic and endocrine control of renin activity in the submaxillary gland of the mouse

Basal activity of submaxillary gland (SMG) renin is high in female mice that carry the Rnr ^s allele and is induced to higher levels by treatment with dihydrotestosterone (DHT). To determine whether the difference in basal activity between high (Rnr ^s/Rnr^s) and low (Rnr ^b/Rnr^b) strains is due to enhanced sensitivity of Rnr ^s/Rnr^s strains to endogenous androgen, we first studied the effect of several types of endocrine ablation on SMG renin in young female mice, and second, we removed normal androgen receptor protein by introducing the X-linked Tfm gene. Adrenalectomy with or without castration had no effect on basal SMG renin; hypophysectomy decreased basal renin activity 400-fold but did not abolish responsiveness to DHT. Loss of androgen receptor did not affect basal renin activity but did prevent enhancement by DHT. Basal and induced renin activities in L.AKR(Alll)/Cy, a congenic strain homozygous for Rnr ^s introduced from AKR/J into the background of C57L/J, an Rnr ^b/Rnr^b type strain, are intermediate between levels observed in the original strains. We conclude that (1) the basal level of SMG renin is regulated directly or indirectly by some pituitary hormone(s) but not by androgen, (2) androgen induction of renin activity requires a normal androgen receptor, and (3) major gene(s) that regulate basal as well as induced SMG renin are in a circumscribed region of chromosome 1.This work has been aided by Grants GM26414 and AM03892 from the National Institutes of Health, a grant from the Texas affiliate of the American Heart Association, and by research contract NO1-CP33255 from the Division of Cancer Cause and Prevention, the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

Increase in liver acid lipase of thyroidectomized rats by thyroid hormones and its inhibition by actinomycin D

Acid lipase activity in the livers of thyroidectomized rats is increased by administration of L-thyroxine. The response is dose-dependent and can be demonstrated within 12 h after treatment. L-Triiodothyronine also evokes a rapid increase in acid lipase activity, and this increase can be inhibited by co-administration of actinomycin D.     

Quantitative changes in potassium,sodium, and calcium in the submaxillary salivary gland and blood serum of rats exposed to 2880-MHz microwave radiation

Na⁺, K⁺, and Ca²⁺ concentrations in the blood serum and submaxillary salivary gland (SSG) were investigated in adult, male rats exposed to 2880-MHz microwaves modulated with 1.5-μs pulses at a pulse repetition rate of 1000 Hz or in a hyperthermal environment. Rats were exposed, one at a time, for 30 min to microwaves producing a specific absorption rate (SAR) of: 4.2, 6.3,6.8,8.4, 10.8, or 12.6 W/kg, or were sham exposed under similar environmental conditions. In a second series, one group of rats was exposed singly for 15, 30, or 60 min to microwaves producing an SAR of 9.5 W/kg and other rats were exposed for similar periods at 40 °C; and 10 rats were sham exposed. Flame photometric analysis indicated that the thresholds of microwave radiation required to induce a change in Na⁺, K⁺, and Ca²⁺ concentrations in the salivary glands are 6.8, 6.8, and 6.3 W/kg, respectively. The directions of Na⁺, K⁺, and Ca²⁺ ion shifts in exposed rats' salivary glands are similar, whether affected by microwaves or hyperthermia. Greater changes in Na⁺ and K⁺ concentrations in SSG of rats exposed to microwaves for 15 and 30 min were found than in those exposed at 40 °C. On the other hand, exposure to hyperthermia at 40 °C or to microwaves for 1 h caused Na⁺ concentration to be increased by 68.7 and 59.5% and K⁺ concentration to be decreased by 29.6 and 21.7%, respectively.     

Evidence for a role of the cytoskeleton in the in vitro folliculogenesis of the thyroid gland of the fetal rat

Summary Thyrotropic hormone (TSH) or cAMP accelerate the formation of follicular cavities in the explanted thyroid gland of the 15-day-old rat fetus. Cytochalasin B or vinblastine and nocodazole or colchicine, which disorganize microfilamental and microtubular structures respectively, inhibit or completely block in vitro-induced folliculogenesis. Exposure of the thyroid tissue to lumicolchicine, a structural isomer of colchicine deprived of antimicrotubular activity, does not inhibit the activation of folliculogenesis induced by TSH. These results are strong evidence for the supposition that microfilaments and microtubules are involved in the TSH-stimulated mechanisms resulting in thyroid folliculogenesis. Folliculogenesis requires the integrity of both microfilaments and microtubules.