A dominant negative CREB (cAMP response element-binding protein) isoform inhibits thyrocyte growth, thyroid-specific gene expression, differentiation, and function.

cAMP mediates the effects of TSH by regulating thyroid follicular cell proliferation, differentiation, and function. To assess the functional importance of the cAMP response element binding protein (CREB) in thyroid follicular cell regulation in vivo, we targeted the expression of a dominant negative (DN) CREB isoform to the thyroid glands of transgenic mice using a tissue-specific promoter. Transgenic mice exhibited severe growth retardation and primary hypothyroidism. Serum levels of TSH were elevated 8-fold above normal levels, and T4 and T3 levels were low. Histologically, the mutant thyroid glands were characterized by poorly developed follicles that were heterogeneous in size with diminished colloid. Ciliated thyroid epithelial cells were observed in the transgenic thyroid glands, suggesting a failure of follicular cell differentiation. Consistent with this hypothesis, the DN CREB transgene inhibited the expression of an array of genes including thyroglobulin, thyroperoxidase, and the TSH receptor in semiquantitative RT-PCR experiments. Altered expression of the thyroid transcription factors Pax-8, TTF-1, and TTF-2 was also observed. These results demonstrate a critical role for CREB in thyroid growth, differentiation, and function in vivo.

Estradiol increases proliferation and down-regulates the sodium/iodide symporter gene in FRTL-5 cells.

Goiter (increased thyroid gland size) is more prevalent in women than men, even in areas where iodine levels in the diet are sufficient. We investigated a possible role of estrogen on thyroid follicular cell growth using rat FRTL-5 thyroid follicular cells as a model. Estrogen receptor-alpha (ERalpha) messenger RNA was present in FRTL-5 cells using a RT-PCR assay and was confirmed by Western blot analysis. An estrogen-responsive reporter gene was transfected into FRTL-5 cells to test the functionality of the endogenous ERs. Estradiol increased the activity of the reporter gene, and the antagonist, ICI182780, inhibited ER-dependent transcription. To extend this analysis, we examined the effect of estradiol on FRTL-5 cell growth. Estradiol increased FRTL-5 cell growth in a time- and concentration-dependent manner in either the absence or presence of TSH. Because iodine is known to inhibit thyroid cell growth, the effect of estradiol on the expression of the sodium/iodide symporter (NIS) was assessed as a potential target of estrogen action. Estradiol blocked TSH-induced NIS expression, and treatment of cells with estradiol and ICI182780 restored TSH-induced NIS expression to normal levels. These data demonstrate that FRTL-5 cells contain functional ERs that enhance cell growth and inhibit expression of the NIS. The demonstration of a direct effect of estradiol on thyroid follicular cells raises the possibility that it may play a role in the sexually dimorphic prevalence of goiter.

A novel mutation (Q40P) in PAX8 associated with congenital hypothyroidism and thyroid hypoplasia: evidence for phenotypic variability in mother and child.

Congenital hypothyroidism associated with thyroid hypoplasia can be caused by several genetic defects, including mutations in the TSHbeta-subunit, the TSH receptor, the G(s)alpha-subunit, and the transcription factor PAX8. Four girls with sporadic congenital hypothyroidism and hypoplastic thyroid glands were analyzed for mutations in PAX8 and TTF2 (FKHL15). Mutations in the coding region of the TSHbeta-subunit gene, the TSH receptor gene, and exons 8 and 9 of G(s)alpha had been excluded previously. Serum TSH concentrations were 150 mU/liter or more, TG levels were within normal limits, and thyroid autoantibodies were absent. Technetium scintigraphies did not reveal the presence of thyroid tissue, but ultrasonography documented hypoplastic, normally located glands. One patient was found to harbor a heterozygous transversion 119A-->C in exon 3 of PAX8 replacing a conserved glutamine by proline in the paired box domain (Q40P). Analysis of her family members revealed that her mother, who has a thyroid gland of normal size and mild, adult-onset autoimmune hypothyroidism, is also heterozygous for this mutation. Functional analyses of the PAX8 Q40P mutation showed impaired binding to a PAX8 response element and absent trans-activation of a thyroid peroxidase promoter luciferase reporter gene. These findings confirm the important role of PAX8 in the development of the thyroid, but they indicate that PAX8 gene mutations may have a variable penetrance or expressivity. The absence of mutations in the coding sequences of the analyzed genes in the three other patients supports the concept that the pathogenesis of congenital hypothyroidism associated with thyroid hypoplasia is diverse.

Structural analysis of the thyrotropin receptor in four patients with congenital hypothyroidism due to thyroid hypoplasia.

Sporadic congenital hypothyroidism is most commonly caused by developmental abnormalities of the thyroid gland. More rarely, it is due to defects in gene products involved in the regulation of the hypothalamic-pituitary-thyroid axis or thyroid hormone synthesis. Loss of function mutations in the thyrotropin (TSH) receptor have been shown to result in resistance to biologically active TSH. In complete resistance to TSH, the thyroid gland is hypoplastic and unable to synthesize and secrete sufficient amounts of thyroid hormones. In partial resistance, referred to as euthyroid hyperthyrotropinemia, the size of the gland and the thyroid hormone levels are normal at the expense of an elevated TSH. Four patients with sporadic congenital hypothyroidism and properly located hypoplastic thyroid glands were included in this study. Serum TSH concentrations were 150 mU/L or higher, serum thyroglobulin levels were within normal limits (6.1 to 8.2 ng/mL; normal range: 2.1 to 32 ng/mL), and thyroid autoantibodies were absent. The coding region of the TSHbeta subunit gene, the TSH receptor gene, and exons 8 and 9 of Gsalpha were analyzed by direct sequencing and found to be normal in all patients. One patient was heterozygous for a G to A transition in the TSHbeta gene resulting in a substitution of alanine by threonine at position -7 of the signal peptide. This substitution was also found in her euthyroid father. In addition, Southern analysis of the TSH receptor gene excluded major structural alterations. These findings support previous reports that indicate that TSH resistance is genetically heterogeneous. In addition to mutations in the TSH receptor or the Gsalpha genes, other genetic defects can lead to an identical phenotype. These observations also suggest that TSH receptor mutations might be a relatively rare cause of congenital thyroid hypoplasia.

Treatment of pyoderma gangrenosum with benzoyl peroxide.

A fifty-three year old woman presented with pyoderma gangrenosum on the right buttock. She had associated intestinal symptoms, but repeated roentgenologic studies revealed no abnormalities. Local treatment with benzoyl peroxide (20 percent) lotion produced clearing of the cutaneous lesion in about six weeks.

Reactions to manganese and cadmium in tattoos.

Concomitant allergic reactions to manganese and cadmium sulfide developed in a patient in tattooed areas. Although the reaction to cadmium was elicited by sunlight, attempts to reproduce it with artificial long-wave ultraviolet light were unsuccessful.

Mathematical relationships between the intake of n-6 and n-3 polyunsaturated fatty acids and their contents in adipose tissue of growing pigs.

To establish the relationships between the fatty acid composition of adipose tissue in growing pigs and the intake of fatty acids, we performed a feeding trial and did a literature survey. Six groups of pigs were fed diets with variable combinations of corn, linseed and fish oil. After 38 days, biopsies of adipose tissue were analyzed for their contents of linoleic, α-linolenic, eicosapentaenoic and docosahexaenoic acid. For the four fatty acids, intake data and adipose tissue levels were also collected from the literature. Linear correlations were computed for the intake of each polyunsaturated fatty acid and its level in adipose tissue, the data set consisting of either the original results only or combined with literature figures. The observed strong correlations between dietary and fat tissue polyunsaturated fatty acids indicate that the fatty acid composition of the diet may be used as an index of the fatty acid composition of the diet, and vice versa. The regression equations presented can be used to steer the fatty acid composition of adipose tissue of growing pigs by the fatty acid composition of their diet.

Intake of essential fatty acids by growing-finishing pigs kept on smallholdings in central Vietnam.

The intake of linoleic acid (LA) and alpha-linolenic acid (ALA) in relation to average daily weight gain (ADG) was studied in growing-finishing pigs kept on smallholdings in Central Vietnam. Groups of three piglets each were assigned randomly to 12 farms, where they were fed on local feedstuffs according to the farmer's choice but were given a restricted amount of dry matter according to a preset feeding regimen. On arrival at the farms, the pigs weighed 10.4 +/- 0.9 kg (mean +/- SD, n = 36) and at 130 days of age they weighed 45.4 +/- 9.6 kg. Dietary LA concentration ranged from 1.34 to 2.41 g/MJ metabolizable energy (ME) and ALA from 0.06 to 0.33 g/MJ ME. On a farm level, dietary LA and ALA concentrations were significantly correlated with their concentrations in adipose tissue, both correlation coefficients being 0.63. Dietary protein concentration and protein:energy ratio were significantly correlated with ADG, the correlation coefficients being 0.68 and 0.64. For individual piglets there were significant correlations between either LA or ALA in adipose tissue and ADG, the correlation coefficients being 0.37 and 0.45. Dietary protein concentration or protein:energy ratio was correlated with the dietary contents of LA and ALA. It is uncertain whether LA and ALA intake had a causal relationship with ADG. Since dietary LA levels were above the LA requirement, LA intake may not have limited growth. It is suggested tentatively that, through enhanced disease resistance, supplementation with ALA of the diets on the farms studied might have a positive influence on ADG.

Inhibition of transcription factors by anti-inflammatory and anti-rheumatic drugs: can variability in response be overcome?

1. The drugs used in the treatment of rheumatoid arthritis (RA) form a diverse group with unpredictable adverse effects, mostly weak efficacy and variable responses. Despite their differences, a common feature of many anti-inflammatory and disease-modifying anti-rheumatic drugs (DMARD) is inhibition of pro-inflammatory transcription factors, particularly nuclear factor (NF)-kappaB and activator protein (AP)-1. 2. The present brief review identifies those drugs capable of inhibiting transcription factors, particularly steroids, gold salts, D-penicillamine, cyclosporine A and possibly salicylates. 3. The newer biological inhibitors of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta are capable of indirect inhibition of NF-kappaB activation, although even with these potent agents the problem of variability in response has not disappeared. 4. The development of selective inhibitors of the transcription factor NF-kappaB should have the benefit of the anti-inflammatory drugs and DMARD, both new and old. 5. It is hypothesized that this strategy will overcome much of the variability in the therapeutic response and adverse effects that limit the usefulness of the existing drugs in the treatment of RA.

Synovial fluid induced nuclear factor-kappaB DNA binding in a monocytic cell line.

OBJECTIVE: To determine the effects of synovial fluids (SF) on DNA binding activity of transcription factor nuclear factor-kappaB (NF-kappaB) in the Mono Mac 6 monocytic/macrophage cell line as a model for the interaction between SF and synovial tissue macrophages in arthritis. METHODS: Mono Mac 6 cells were incubated with SF from the knee joints of human subjects with rheumatoid arthritis (RA), undifferentiated seronegative oligoarthritis, and osteoarthritis (OA). Nuclear extracts prepared from the Mono Mac 6 cells and RA synovial tissue were analyzed by electrophoretic mobility shift analysis (EMSA) for NF-kappaB DNA binding proteins. RESULTS: Induction of NF-kappaB DNA binding by the p65(RelA)/p50 heterodimer was observed in response to incubation of Mono Mac 6 cells with SF (20% in culture medium) from 5 of 8 subjects with RA, 4 of 5 with OA, and none of 3 with undifferentiated seronegative oligoarthritis. Incubation of SF with neutralizing antibodies against tumor necrosis factor-alpha (TNF-alpha), but not antibodies against interleukin 6 (IL-6), significantly reduced the induction of p65/p50 binding activity in SF from subjects with RA and OA. Unexpectedly, a slowly migrating SF inducible NF-kappaB-binding complex was observed in EMSA of Mono Mac 6 cells after incubation with SF from 5 of 8 RA and 2 of 5 OA subjects. The induction of this complex by SF was not affected by neutralization of TNF-alpha or IL-6 in SF, and the complex was not inducible by TNF-alpha, IL-1beta, TNF-alpha/IL-1beta, IL-6, platelet derived growth factor, lipopolysaccharide, or tetradecanoyl phorbol acetate. The slowly migrating complex could not be supershifted with antibodies against NF-kappaB, Jun, or the transcriptional coactivators p300 or CBP. A NF-kappaB-binding complex with similar slow mobility was observed in nuclear extracts prepared from fresh human RA synovial tissue. CONCLUSION: Biological activity of TNF-alpha in SF from RA and OA subjects is capable of inducing p65/p50 NF-kappaB DNA binding activity in macrophages. A property of SF that is independent of TNF-alpha and other cytokines is responsible for the induction of a novel slowly migrating NF-kappaB-binding complex. Soluble mediators in SF of subjects with RA and OA can therefore modulate binding of nuclear proteins to the NF-kappaB binding site in macrophages and may play a role in inflammatory gene expression in arthritis.

Influence of dietary linseed, fish and coconut oil on growth performance of growing pigs kept on small holdings in central Vietnam.

The aim of the study was to investigate the effect on growth performance of the addition of 5% of either linseed, fish or coconut oil to the diet of growing pigs kept on small holdings in central Vietnam. The diets were fed on six different farms; there were three animals per treatment per farm. The farmers fed a base diet according to personal choice, but were instructed to use linseed, fish and coconut oil. The diets were fed to the pigs from 70 to 130 days of age. The diets with 5% linseed oil on average contained 2.4 g alpha-linolenic acid/MJ of metabolizable energy (ME) and the diets with 5% fish oil on average contained 0.06 and 0.75 g eicosapentaenoic and docosahexaenoic acid/MJ ME, respectively. The relative percentages of docosahexaenoic, eicosapentaenoic, alpha-linolenic and linoleic acid in adipose tissue were determined by the intake of the corresponding fatty acids. Dietary linseed oil vs. coconut oil significantly enhanced daily weight gain and vs. fish oil it also stimulated growth, but this effect just failed to reach statistical significance. It is concluded that extra intake of alpha-linolenic acid may stimulate growth in growing-finishing pigs, this effect being independent of the conversion of alpha-linolenic acid into eicosapentaenoic and docosahexaenoic acid.

Feeding of spinach or sweet-potato leaves and growth performance of growing pigs kept on smallholder farms in Central Vietnam.

We investigated whether the addition of either spinach or sweet-potato leaves to the diet of growing pigs, kept in small holdings in Central Vietnam, would improve growth performance. A control diet was formulated and mixed with each of the vegetables to a final concentration of 15% of the total dietary dry matter. The diets were fed to the pigs from 70 to 100 days of age on six different smallholder farms in Central Vietnam. There were three animals per treatment group per farm and biopsies of adipose tissue were analysed for their contents of alpha-linolenic, eicosapentaenoic and docosahexaenoic acids. The diets without and with vegetables contained on average 0.14 and 0.32 g alpha-linolenic acid per MJ metabolizable energy. The relative percentage of alpha-linolenic acid in adipose tissue was raised by the intake of the vegetables. Eicosapentaenoic acid was not detectable in adipose tissue and the level of docosahexaenoic acid was unchanged. There was a significant stimulatory impact of the intake of either spinach or sweet-potato leaves on growth performance of the growing pigs. It is suggested that an adipose tissue content of alpha-linolenic acid less than 1% of total fatty acids does not allow maximum growth in growing pigs.

Validation of cleaning procedures for highly potent drugs. I. Losoxantrone.

The validation of a procedure designed to clean glass and stainless steel surfaces after exposure to the experimental anticancer drug losoxantrone is described. The cleaning procedure, using water and hypochlorite bleach, was validated using a wipe test and a high-performance liquid chromatography (HPLC) method developed to quantitate low levels of losoxantrone. The HPLC method is shown to be linear and reproducible (relative standard deviation (RSD): 7.1% or less), with a detection limit of 2 ng/ml. Recoveries of 71.0%, 50.1%, and 57.6% were reproducibly obtained from the wipe pads, glass plates, and stainless steel plates, respectively, at levels of 70-140 ng per 100 cm2. The cleaning procedure is shown to clean glass and stainless steel plates to less than 20 ng and 17 ng losoxantrone per 100 cm2, respectively. These results demonstrate the need to fully characterize the recovery of drugs from surfaces and swabs in order to properly validate cleaning procedures.