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We report the structural properties, infrared (IR) and Raman spectra, dipole moment, polarisability, hardness and chemical potential of the trans and cis configurations of 4-hydroxyazobenzene calculated using the B3LYP functionals. All calculations were performed with the following basis sets: 6-31G, 6-31++G, 6-31G(d,p), 6-31++G(d,p), 6-31G(2d,2p), 6-31++G(2d,2p) and 6-311++G(2d,2p). We observed that 6-31++G(d,p) gives similar results to 6-311++G(2d,2p). Consequently, SVWN and PW91 methods were also used in association with 6-31++G(d,p) to test the influence of the different models of exchange and correlation functionals. A planar structure was obtained for all the optimised trans configuration structures. In both isomers, the presence of the hydroxyl group leads to an asymmetry in certain structural parameters. From these results, two IR or Raman active frequencies can be used to easily distinguish trans and cis configurations. The trans configuration was found to be more stable than the cis configuration by 67 +/- 2 kJ mol(-1) at 0 K. The difference of the dipole moment between trans and cis for 4-hydroxyazobenzene was found to be lower than for trans and cis azobenzene.

Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. We addressed the presence of apoptosis in muscle from patients with mitochondrial DNA (mtDNA) disorders. Taking advantage of the mosaic pattern of muscle morphological anomalies associated with heteroplasmic mtDNA alterations, we have used an in situ approach to address the relationship between apoptosis and respiratory defect, mitochondrial proliferation and mutation load. Different patterns of mitochondrial morphological alterations were provided by the analysis of muscles with large mtDNA deletion (16 cases) or with the MELAS mutation (4 cases). The patient's age at biopsy ranged from 0.4 to 66 years and the muscle mutant mtDNA proportion from 32 to 82%. Apoptotic muscle fibres were observed in a small proportion of muscle fibres of 16 out of the 20 biopsies by three different detection methods for different steps of apoptosis: caspase 3 activation, fragmentation of nuclear DNA [terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay] or overexpression of the pro-apoptotic factor Bax. Analysis of apoptotic features in parallel to cytochrome c oxidase (COX) and succinate dehydrogenase activity of more than 34,000 individual muscle fibres showed that apoptosis occurred only in muscle fibres with mitochondrial proliferation (ragged red fibres, RRF) irrespective of their COX activity. Molecular analyses of single muscle fibres evidenced that, as expected, the presence of COX defect was associated with higher proportion of mutant mtDNA and lower amount of normal mtDNA. Within COX-defective fibres, the presence of mitochondrial proliferation was associated with increase of the mtDNA content but without change in the ratio between normal and mutant mtDNA molecules, thus showing that mitochondrial proliferation was accompanied by similar amplification of normal and mutant mtDNA molecules. Within RRF, apoptosis was associated with higher mutation proportion, suggesting that it was provoked by severe respiratory defect in the same time as increased mitochondrial mass. In conclusion, apoptosis most probably contributes to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and high mutation load. When considering training therapeutics, one will have to take into account the possibility to induce apoptosis in parallel to mitochondrial proliferation.

Background: The aim of this study was to identify factors predictive of survival after curative resection of hepatocellular carcinoma (HCC) in noncirrhotic liver., Study Design: Eighty-four patients underwent resection of HCC in noncirrhotic liver between January 1998 and December 2003. Univariate and multivariable analyses were used to retrospectively identify factors associated with overall survival and disease-free survival when resection was curative for the primary tumor., Results: Overall 1-, 3-, and 5-year survival rates were 77.8%, 55.0%, and 44.4%, respectively, and 84.0%, 62.0%, and 50.0% when resection was curative for the primary tumor. HCC recurred in 27 patients (39.1%). Recurrence was intrahepatic in 14 patients (51.9%), extrahepatic in 3 patients (11.1%), and both intra- and extrahepatic in the remaining 10 patients (37.0%). In multivariable analysis, three independent factors were associated with poorer overall survival and recurrence-free survival, namely multiple tumors, macroscopic vascular invasion, and nonuse of adjuvant iodine-131-iodized oil., Conclusions: Aggressive operation is an effective treatment for HCC in noncirrhotic patients, whatever the degree of liver fibrosis. Multiple tumors and macroscopic vascular invasion are poor prognostic factors. Postoperative iodine-131-iodized oil injection appears to prevent recurrence and improve overall survival, although this needs to be confirmed in a prospective randomized trial.

We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA(Asn) gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA(Asn) and cause a fatal mitochondrial disease.

A cell line of functional normal human adult thyroid cells was isolated 3 years ago. This cell line was used as a model to study human thyroglobulin (hTgb) production quantified in 5% fetal calf serum Click-RPMI medium and in the presence of insulin and thyrotropin (two hormones [2H]) to get maximal hTgb production. In this paper we demonstrate that these cells (line ARAMIS) follow the general dualistic opposition between growth and differentiation. Thyroglobulin cell production in 2H-stimulated cells is dependent on cell density and is not constant with time of culture in growing and in quiescent cells. High serum levels required for cell proliferation antagonize thyroglobulin cell production. Furthermore, in the absence of cell proliferation, the longer the cells stay in the stationary phase in 2H-medium, the better they produce thyroglobulin with time. The longer the cells stay in the stationary phase in 1H (insulin) medium, the higher will be the total thyroglobulin production and the initial rate in thyroglobulin production after TSH addition. The longer the cells stay in the stationary phase in 1H (insulin) medium, the higher are cyclic adenosine monophosphate (cAMP) levels after thyrotropin (TSH) stimulation. This is observed both for initial rates and total production. Neither insulin nor cell-cell interactions occurring during the stationary phase modify basal cAMP levels. Altogether the data demonstrate that in cell culture conditions that exclude proliferation, a gain in TSH sensitivity appears versus time in insulin-stimulated quiescent normal human thyroid cells during their stationary phase. This improved differentiating status appears to be TSH- and cAMP-independent. It could be an insulin or insulin-like growth factor-1 (IGF-1)-dependent trophic effect promoting an increase in TSH-receptor number or sensitivity. But once again and as mentioned previously, we cannot exclude from the data that cell to cell interactions between silent and "informed" cells (in other words, autocrine phenomena) could result, with time, in the recruitment of silent resting cells, explaining the gain in TSH sensitivity.

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl-N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.

Background: The age-related decline of dehydroepiandrosterone and its sulfate ester levels is thought to be related to the development of age-associated usual modifications, such as neuromuscular function impairments. It is often claimed that individuals can enhance their muscular capacity by boosting dehydroepiandrosterone levels through oral supplementation. However, to our knowledge, there have been no controlled studies on a significant number of individuals demonstrating positive effects on the neuromuscular system. This study determines if 1-year administration of a replacement dose of dehydroepiandrosterone, 50 mg/d, orally administered, could have a beneficial influence on several determinants of the muscular function altered during aging., Methods: This work was completed within the frame of the DHEAge Study, which was conducted in France from March 1, 1998, to October 31, 1999. It was performed on 280 healthy ambulatory and independent men and women aged 60 to 80 years. The study design was a double-blind placebo-controlled trial. Dehydroepiandrosterone sulfate serum concentration, handgrip strength, isometric and isokinetic knee muscle strength, and thigh (fat and muscle) cross-sectional area were analyzed before and just after 12 months of placebo or dehydroepiandrosterone treatment., Results: The results give evidence that dehydroepiandrosterone administration restores dehydroepiandrosterone sulfate serum concentrations to the normal range for young adults (aged 20-50 years). However, no positive effect inherent to dehydroepiandrosterone treatment was observed either on muscle strength or in muscle and fat cross-sectional areas., Conclusions: The compensation of the deficit of dehydroepiandrosterone during aging using a 50-mg/d dose does not induce beneficial effects on muscle state in healthy subjects. The conditions in which dehydroepiandrosterone could contribute to preserve or improve muscle strength and morphological features still need to be determined.

Photodynamic therapy with Verteporfin, a potent photosensitizer dye, is a very effective treatment for age related macular degeneration due to choroidal neovascularization. Photodynamic therapy offers the potential for selective tissue injury in part attributable to preferential localization of Verteporfin, administrated by intravenous infusion, to the choroidal neovascularization complex and irradiation of the complex with non-laser thermal light at 690 nm resulting in at least temporary thrombosis and vessel closure. Verteporfin is a benzoporphyrin derivative monoacid ring A formulated as a unilamellar liposome. In the blood Verteporfin is associated with lipoprotein fractions and is rapidly cleared via a receptor-mediated uptake mechanism due the high expression of LDL receptors in neovascular tissues. Verteporfin was undetectable in plasma 24 hr after infusion of the recommended dose: 6 mg/m2 of body surface area. The main side effect is photosensitivity of skin which is usually short-lived (24-48 hr) with a low incidence (2.3%). As skin photosensitivity depends on circulating rather than tissue drug levels, we investigate the possibility of developing a simple, fast and reliable spectrofluorometric method to measure plasma Verteporfin levels. Fluorescence emission spectrum (550-750 nm) of 1:10 saline diluted plasma with lambda exc=430 nm showed a characteristic emission peak at 692 nm, the height being proportional to the Verteporfin levels. The sensitivity is around 100 ng/ml and the pharmacokinetics of Verteporfin has been studied from 0 to 5 hr after infusion in six patients older than 65 years with age-related macular degeneration.

Obstructive sleep apnoea syndrome (OSAS) induces marked haemodynamic fluctuations during sleep that might be deleterious to the cardiovascular system. The influence of daytime blood pressure (BP) levels and aging on short-term BP variability during sleep in OSAS patients was investigated. Twenty-nine subjects with newly-diagnosed untreated OSAS were categorised into three groups: normotensive subjects aged <50 yrs (n=10); subjects aged <50 yrs with untreated hypertension (n=8); and normotensive subjects aged >50 yrs (n=11). Beat-by-beat BP was recorded with a Finapres device during polysomnography. The average values+/-SD of apnoea-related BP elevations and the values of the frequency distribution of all BP variations during sleep were assessed to estimate short-term BP variability. Apnoea-related systolic (or diastolic) BP elevations were significantly greater in hypertensives than in normotensives aged <50 yrs (50.3+/-4.88 versus 30.7+/-2.14 mmHg, p<0.001), as was the SD of systolic (or diastolic) BP variations during sleep (19.6+/-2.22 versus 11.1+/-0.73, p<0.001). Short-term BP variability was not significantly increased in normotensive elderly patients. To conclude, the results suggested that systemic hypertension is associated with a greater exacerbation of short-term variability during sleep in obstructive sleep apnoea syndrome patients.

The long-term culture of functional follicular cells from normal adult human thyroid tissue has been obtained. They were expanded using a 1:2 split ratio until passage 28 (present status) in Click-RPMI medium enhanced with 5% fetal calf serum and diverse associations of hormones or components including porcine insulin and bovine thyrotropin. At passages 10 and 20, chromosome countings showed a normal diploid number and a normal karyotype. In calf serum containing media, cells are epithelial in the presence of thyrotropin (TSH) but present a slight elongated form in the absence of TSH. In serum-free media, 30 minutes after TSH stimulation, both epithelial and elongated cells changed in morphology to stellate-shaped, arborized forms, indicating the presence of functional TSH-receptors even in long term (18 months) TSH-free cultures. Cells produce thyroglobulin constitutively and large amounts of thyroglobulin are easily recovered in TSH-supplemented media, especially in the presence of insulin. Thyroglobulin production was increased versus days under TSH or insulin stimulation. Combination of the two hormones clearly resulted in a synergistic and not an additive effect. The other hormones present in the 6H components (transferrin, glycylhistidyl-lysine, somatostatin, and hydrocortisone) had no positive effect on thyroglobulin accumulation in media in our experimental conditions. Addition of TSH to hormone-free cultures or to insulin-, insulin plus hydrocortisone-, or 5H-containing cultures resulted in a clear increase in thyroglobulin production. Withdrawal of TSH from 6H cultures resulted in a decrease in thyroglobulin accumulation in media. Six months were required to select fibroblast-free cultures and to get passage 6. But only 17 months separated passage 6 to passage 28, indicating that the proliferative rate is increasing with in vitro cell adaptation. Such normal adult thyroid cells, thyroglobulin-producing, TSH, and insulin-sensitive, represent a new normal human thyroid cell line allowing comparative studies with cells originating from pathologic thyroid tissues.

Although mitochondrial DNA deletions have been shown to accumulate in cytochrome c oxidase deficient muscle fibres of ageing muscle, this has not been demonstrated for point mutations. In this study, we investigated the occurrence of mitochondrial DNA alterations (point mutations and deletions) in cytochrome c oxidase deficient muscle fibres from 14 individuals, without muscle disease, aged 69-82 years. Immunohistochemical investigation showed that the majority of the cytochrome c oxidase deficient muscle fibres expressed reduced levels of subunit II of cytochrome c oxidase, which is encoded by mitochondrial DNA, whereas there was normal or increased expression of subunit IV of cytochrome c oxidase, which is encoded by nuclear DNA. This pattern is typical for mitochondrial DNA mutations causing impaired mitochondrial translation. Single muscle fibres (109 cytochrome c oxidase deficient and 109 normal fibres) were dissected and their DNA extracted. Mitochondrial DNA point mutations were searched for in five tRNA genes by denaturing gradient gel electrophoresis while deletions were looked for by polymerase chain reaction amplification. High levels of clonally expanded point mutations were identified in eight cytochrome c oxidase deficient fibres but in none of the normal ones. They included the previously described pathogenic tRNALeu(UUR)A3243G and tRNALysA8344G mutations and three original mutations: tRNAMetT4460C, tRNAMetG4421A, and a 3-bp deletion in the tRNALeu(UUR) gene. Four different large-scale mitochondrial DNA deletions were identified in seven cytochrome c oxidase deficient fibres and in one of the normal ones. There was no evidence of depletion of mitochondrial DNA by in situ hybridisation experiments. Our data show that mitochondrial DNA point mutations, as well as large-scale deletions, are associated with cytochrome c oxidase deficient muscle fibre segments in ageing. Their focal accumulation causes significant impairment of mitochondrial function in individual cells in spite of low overall levels of mitochondrial DNA mutations in muscle.

Six bitches free of canine herpesvirus 1 (CHV-1) were vaccinated against the virus; a first injection was given 10 days after the presumed date of mating and a second six weeks later. Six similar bitches were left unvaccinated as controls, and all the pups were challenged oronasally with a virulent strain of CHV-1 at three days of age. All the vaccinated bitches seroconverted and had high antibody titres when the puppies were challenged, but the control bitches remained seronegative. In the control group, 62 per cent (18 of 29) of the pups died of CHV-1-induced disease; most of them showed typical clinical signs and macroscopic lesions, and CHV-1 infection was confirmed by the isolation of the virus or by PCR. None of the puppies in the vaccinated group died of CHV-1 infection. The efficacy of the vaccine was confirmed in CHV-1-positive breeding units. The rate of pregnancy tended to be higher in vaccinated bitches and the mortality of pups before weaning was significantly reduced in the litters born to vaccinated bitches.

Many different pathogenic mutations in the mitochondrial (mt) transfer RNA (tRNA) genes have been reported for patients with mitochondrial encephalomyopathy. Although some of them are recurrent, most have only been described once and appear to be restricted to one patient or to one family. The incidence of mt tRNA gene alterations is not known, even though the frequency of some recurrent mutations has been analysed both in patients and in the general population. In this study, we describe the results of stepwise screening for sequence variations in the mt tRNA genes of 166 patients selected according to several criteria. Extensive sequence analysis of the tRNA genes was performed using denaturing gradient gel electrophoresis. A total of 31 patients (19%) were found to harbour significant levels of a pathogenic mutation, thus confirming the importance of mt tRNA mutations in human pathology. Forty-three different sequence variations were found, illustrating the great diversity of the mtDNA sequence in humans. The functional assessment of all these sequence variations represented a difficult task; it was mostly based on indirect data, such as the phylogenetic conservation of modified nucleotides and the proportions of variant species in different tissues of the index case or in blood of maternal relatives. Direct demonstration of a correlation between the proportion of heteroplasmic sequence variations and the cytochrome c oxidase defect was performed at the single muscle-fibre level. Eleven heteroplasmic sequence variations were found, six of which are new mutations. One is a known Caucasian polymorphism but the other 10 are pathogenic. Two of them are the well-known pathogenic MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (A3243G) and MERRF (myoclonic epilepsy with ragged-red fibres) (A8344G) point mutations. They were found in 23 patients. The eight other mutations were restricted to one patient. The pathogenic nature of these mutations was demonstrated directly for five of them and hypothesized from indirect arguments for the other three. Thirty-two homoplasmic sequence variations were found. Twenty-nine were considered to be polymorphisms, even though 15 of these were found for the first time in our patients and two others had been reported previously as pathogenic. The pathogenic nature of three homoplasmic variants remains questionable.

A quantitative analysis of deltoid muscle biopsy specimens was performed by light microscopy in 26 males and 25 females aged 50-79 years without known neuromuscular disease. Muscle fibre size, fibre type distribution and increase in mitochondrial content in muscle fibres were examined using a semi-automatic image analysis system. This study showed significant age- and gender-related differences. In females, there was marked atrophy of type II fibres with increasing age, specially of type IIb fibres, but no significant change in muscle fibre type distribution. In males, there was diminution in the relative proportion of type IIb fibres with increasing age but no significant muscle fibre atrophy. Mitochondrial aggregates increased with age, and this increase was observed earlier in females than in males. The gender-related morphological changes observed in the present study differ somewhat from those reported in the literature. The differences between males and females may be partially related to gender differences in muscular activity and may reflect an earlier decline in deltoid muscle strength in females. The existence of these age and gender changes should be taken into account in the interpretation of muscle biopsies of aged individuals.

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.

The polar planar compound hexamethylene bisacetamide (HMBA) is an inducer of terminal differentiation which has been extensively studied in the murine erythroleukemia cells (MELC). We have tested this compound in normal porcine thyroid cells in primary culture where it either activates or inhibits the major tissue specific functions of these cells: it induces the reorganization of cells into follicles, prevents the loss of thyrotropin sensitivity in monolayer cells, activates cell growth but inhibits their iodide metabolism. In this paper, we demonstrate that HMBA acts on the total thyroglobulin levels measured in cell layers plus media. This specific marker of thyroid tissue is increased by HMBA both in kinetics and in concentration-response experiments. HMBA per se does not increase the total cyclic AMP measured either during the first hours after stimulation or in the following days when compared to controls. As expected, cyclic AMP in the same experiment increased rapidly within minutes after the cells were challenged by TSH (positive control). Altogether, the results show that the drug HMBA mimics thyrotropin effects on thyroglobulin levels measured in porcine thyroid cells in culture. This modulation cannot be explained by an increase in cyclic AMP, indicating that despite similarities between TSH and HMBA effects, the mechanism of the mode of action of these two molecules is very different.

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC50 of 5 x 10(-5) M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC50 being also of 5 x 10(-5) M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10(-3) M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10(-3) M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

Neurogenic motor evoked potential (NMEP) monitoring, which basically represents a monitoring of both motor and somatosensory tracts, has been proposed as a warning system in preventing neural damage during spinal surgery. The aim of this study was to report our clinical experience in 112 consecutive patients undergoing surgery for spinal deformity, and to emphasize the interest of NMEP monitoring. NMEPs were elicited in each patient by electrical stimulation of the spinal cord via needle electrodes placed by the surgeon in the rostral part of the surgical field, and recorded from the right and left sciatic nerves. Concomitantly, somatosensory evoked potentials (SSEPs) were obtained using a standard method. No false-negative cases of intra-operative spinal cord damage were reported. In 3 patients, both NMEPs and SSEPs suddenly disappeared during specific surgical manipulations of the vertebral implants. In these patients, the level of the lesion was easily recognized by moving the stimulating electrodes of NMEPs along the spinal cord, allowing the surgeon to perform laminectomy at the appropriate vertebral level. Spinal cord decompression was a success in two patients, the last unfortunately being paraplegic. Two additional patients exhibited transient reduction in NMEP amplitude at the insertion of a rod while SSEPs did not change significantly. In these two cases, the surgeon modified his procedure according to the NMEP changes, possibly avoiding a neurological complication. Each time evoked potentials were significantly altered, significant information was more rapidly acquired with NMEPs than with SSEPs. These results suggest that NMEPs can be used as primary choice for detecting impeding lesion of the spinal cord during critical steps of spinal surgery.

The polar compound hexamethylenebisacetamide (HMBA) is a differentiating agent in the murine erythroleukemia cell system (MELC). It induces, like dimethylsulfoxide, the commitment to terminal differentiation leading to a recovery in the expression of several genes like the globin gene. This molecule which also induces differentiation in other cellular types is a growth agent for human, ovine and porcine thyroid cells. Forty-eight hours after the onset of culture, porcine thyroid monolayer cells do not respond to thyrotropin (TSH). We demonstrate that a pretreatment from the onset of culture with HMBA of porcine thyroid cells prevents the loss of TSH-sensitivity. The TSH-sensitivity is concentration-dependent in HMBA and leads to the reorganization of cells into follicles, even in the presence of HMBA. However, the withdrawal of HMBA when TSH is added is absolutely required to obtain a total recovery in iodide trapping and organification. If HMBA is present during TSH-stimulation, it inhibits iodide trapping partially but iodide organification completely Cells remain sensitive to TSH for at least 12 days if HMBA treated, and their sensitivity is totally restored after 3, 6 or 9 days of TSH-stimulation. HMBA, which is, like TSH, a growth agent for the thyroid cell and an agent that maintains some of the specialized functions, could be a putative candidate to obtain normal human thyroid cell lines.

Background: Most techniques used to monitor spinal cord tracts are sensitive to the effects of anesthesia, particularly to volatile anesthetic agents. The aim of this prospective study was to show that evoked potentials recorded from the peripheral nerves after spinal cord stimulation, so-called neurogenic motor evoked potentials, are resistant to clinical concentrations of isoflurane or desflurane, compared with somatosensory-evoked potentials., Methods: Twenty-three patients were studied during surgery to correct scoliosis. The background anesthetic consisted of a continuous infusion of propofol. Isoflurane (n = 12) or desflurane (n = 11) were then introduced to achieve 0.5 and 1.0 end-tidal minimum alveolar concentrations (MAC), both in 50% oxygen-nitrous oxide and in 100% oxygen. Somatosensory-evoked potentials were elicited and recorded using a standard method, defining cortical P40 and subcortical P29. Neurogenic motor-evoked potentials were elicited by electric stimulation of the spinal cord via needle electrodes placed by the surgeon in the rostral part of the surgical field. Responses were recorded from needle electrodes inserted in the right and left popliteal spaces close to the sciatic nerve. Stimulus intensity was adjusted to produce a supramaximal response; that is, an unchanged response in amplitude with subsequent increases in stimulus intensity. Measurements were obtained before introducing volatile agents and 20 min after obtaining a stable level of each concentration., Results: Isoflurane and desflurane in both 50% oxygen-nitrous oxide and 100% oxygen were associated with a significant decrease in the amplitude and an increase in the latency of the cortical P40, whereas subcortical P29 latency did not vary significantly. Typical neurogenic motor-evoked potentials were obtained in all patients without volatile anesthetic agents, consisting of a biphasic wave, occurring 15 to 18 ms after stimulation, with an amplitude ranging from 1.3 to 4.1 microV. Latency or peak-to-peak amplitude of this wave was not significantly altered with isoflurane and desflurane, either in the presence or in the absence of nitrous oxide., Conclusions: Compared with cortical somatosensory-evoked potentials, neurogenic motor-evoked potential signals are well preserved in patients undergoing surgery to correct scoliosis under general anesthesia supplemented with isoflurane or desflurane in concentrations as great as 1 MAC.

Hexamethylenebisacetamide (HMBA) provokes in murine erythroleukemia cells (MELC) a commitment to terminal differentiation leading to the activation of the expression of hemoglobin. HMBA has been tested also in other cells from colon cancer, melanoma or lung cancer. However it has not yet been tested in the thyroid. We demonstrate in this paper that HMBA in kinetics and concentration-response experiments increases the proliferation of human thyroid cells isolated from Graves'-Basedow patients. It also acts like a growth factor for ovine and porcine thyroid cells, respectively, from the OVNIS line and the ATHOS line. This molecule which is a differentiating factor in the MELC system and a growth factor in human thyroid cell cultures represents a potential to get human thyroid cell lines expressing specialized functions.

A 48-year-old man presented with impaired joint movement and buccofacial apraxia. The disease progressed for six years associating an akineto-hypertonic syndrome, marked anarthria, saliva incontinence, bi-opercular syndrome, bucco-facial apraxia, severe global gestual apraxia and a frontal syndrom. Oculo-motricity and gait were normal. Magnetic resonance imaging of the brain demonstrated fronto-parietal atrophy and HMPAO Tc99 tomography revealed hypoperfusion of the cortex clearly predominating in the left parietal region. These particular findings with predominantly intense joint involvement is similar to the clinical picture in corticobasal degeneration--subcortical signs (progressively uncontrollable hypertonia) together with cortical signs (severe gestual apraxia). The neuroradiological imaging and functional results also suggest a degenerative process.

We report the case of a 64 year old woman treated for 10 years by DOPA for Parkinson's disease and displaying brown urine. Homogentisic acid was found in urine, establishing the diagnosis of alkaptonuria. Clinical and radiological studies demonstrated ochronosis and ochronotic arthropathy, blue pigmentation of ear cartilage and calcification of the intervertebral lumbar disc giving the classical "inverted spine". Interrelation between this metabolic abnormality and Parkinson's disease is discussed. The recent cloning and mapping of the human gene for alkaptonuria to chromosome 3q should bring some clarification among relationships between these two diseases.

Leptomeningeal gliomatosis is a diffuse glial infiltration of the subarachnoid space. It is primary and very rare when primary astrocytoma arises in the leptomeninges from heterotopic neuroglial tissue; it is secondary and more frequently reported when associated with a medullar or cerebral intraparenchymal astrocytoma and secondary involvement of the leptomeninges. Primary and secondary forms are difficult to differentiate before neuropathological examination. The authors report 2 anatomo-clinical cases of leptomeningeal gliomatosis in adults, with clinical courses of 6 months and 40 days respectively. The initial clinical picture was aseptic chronic or subacute meningitis. Cytologic examinations of the cerebrospinal fluid (CSF) showed moderate lymphocytosis, with elevated protein and low glucose levels, without abnormal cells. On case 2 CT scan and in case 1 spinal MRI isolated diffuse meningeal contrast enhancement was present, without intraparenchymal lesion. The neuropathological study revealed a diffuse astrocytoma glial leptomeningeal tumour with a focal involvement of the central nervous system (spinal cord in one case, temporal lobe in the other). In conclusion, an isolated aseptic lymphocytosis meningitis with meningeal abnormal signal may reveal leptomeningeal gliomatosis. Neuropathological examination can distinguish primary from secondary forms.

We report a clinical study of 20 patients aged more than 100 years, and deceased in Charles Foix Hospital, Ivry/Seine, France, where they had stayed for 10 days to 16 years (mean: 3.3 years, median: 4 years). Each of them was studied neuropathologically (the results of this study will be published later on). The case selection excluded only those patients whose neuropathological study could not be complete. The prevalence of sensorial deficits was high: 15/20 patients had deterioration of hearing, 8 of them were deaf; 14/20 patients had deterioration of sight, 4 of them were blind. Motor deficits were numerous: 15 patients were not ambulatory. The patients were classified as demented or not, according to the criteria of the DSMIII R (American Psychiatric Association 1987), on the basis of a retrospective evaluation of clinical records, and of an inquiry among the caring staff. Their mental status was also evaluated by the Global Deterioration Scale of Reisberg et al (1985). Five patients had been demented, four had been intellectually normal, and 11 had suffered from mild disturbances of memory or cognitive functions. This series was not representative of the general population of centenarians, but probably of those institutionalized in France. We observed a low proportion of demented patients despite the prevalence expected from epidemiological studies. This is difficult to interpret. The low proportion of dementia in this small sample is not due to the short duration of the course of diseases responsible for dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of the thyroid follicle is studied in vitro using the porcine thyroid cell line ATHOS. When preformed monolayers of ATHOS cells, grown after more than 50 passages, are cultured in a sandwich of collagen in the presence of thyrotropin (TSH 10 mU/ml) figures, resembling follicles are observed under light microscopy. The mixture forskolin (5 microM) + isobutyl-methyl-xantine (0.25 mM) is as efficient as TSH. From day 2 on, cells form follicle-like structures. These histiotypical figures, that mimic the thyroid architecture, are maintained 4-5 weeks. By electron microscopy, cells appear correctly located inside the follicle-like structure with microvilli facing follicle lumina. Numerous microfollicles and intracellular cavities are also observed. In TSH-free control cell cultures, very small lumina are formed between day 2 and 7. If TSH (10 mU/ml) is acutely added, new large lumina appear in 24 hrs. Removal of TSH or stimulators or addition of calf serum (0.5-10%) results in a complete disorganization of follicles and in the formation of a regular monolayer. Readdition of TSH or stimulators in serum-free medium reinduces the formation of histiotypical figures. This paper indicates that porcine thyroid cells from the ATHOS line even after more than 50 passages in vitro are a suitable tool to study the mechanisms involved in the supraorganization of the thyroid cells into three dimensional follicles.

beta A4 deposits occur in the brain of some individuals over 50 years of age. It could be a part of the aging process or indicate a disease found frequently in the elderly. To address this question, beta A4 immunocytochemistry was performed on the brain of 15 nondemented and 5 demented centenarians, some of whom were affected by Alzheimer's disease. We found beta A4 deposits in the parahippocampal and the superior temporal gyri of all the cases, whatever the clinical state. The hippocampus was frequently spared. The lesion density was not correlated with the severity of the mental deterioration. The constant deposition of beta A4 protein in the brain of very old people indicates that this process does not spare a large proportion of this population. This result favors beta A4 accumulation in the brain being an ineluctable age-related process.

The reconstruction of the thyroid follicle was studied in vitro using the porcine thyroid cell line ATHOS. When performed monolayers of ATHOS cells grown after more than 50 passages are cultured in a sandwich of collagen in the presence of thyrotropin (10 mU/ml), figures resembling follicles are observed under light microscopy. By electron microscopy cells appear correctly polarized, microvilli facing follicle lumina. These histiotypical figures that mimic the thyroid architecture are maintained 4-5 weeks.

Insulin and Insulin-like Growth Factor I (IGF-I) are evaluated for their capacity to affect cell proliferation and plasminogen activator (PA) activity production in an ovine thyroid cell line OVNIS. Insulin at physiological and supraphysiological doses induces cell proliferation and increases PA activity. IGF-I, which is also clearly mitogenic for these cells, surprisingly does not modulate PA activity. The results indicate that the growth promoting effect is mediated through the insulin and IGF-I receptors whereas PA activity is solely regulated via the insulin receptors.

Thyroglobulin secreted by porcine thyroid cells in serum-free culture was previously found (Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293) to contain more highly branched complex carbohydrate chains than the thyroid-derived molecules. When assayed for their ability to react with polyclonal antibodies directed against the natural prohormone in competitive radioimmunoassays, using the porcine antigen as iodinated tracer and standard competitor, in vitro thyroglobulin appeared to be 4-fold less immunoreactive than the in vivo molecule. However, both types of thyroglobulin exhibited superimposable incomplete displacement curves after peripheral deglycosylation using a mixture of neuraminidase, alpha-, and beta-galactosidases while they behave as good competitors as the native antigen after removal of the majority of the carbohydrate chains by Endo-beta-N-acetylglucosaminidase F. Solid-phase binding assays revealed that in vitro thyroglobulin was able to bind less antibodies than its thyroid-derived counterpart before as well as after treatment with exo- or endoglycosidases. Furthermore, only 20% of thyroglobulin biosynthetically labeled with glucosamine could be precipitated with specific antibodies followed by addition of staphylococcal-protein A, whereas up to 61% of the labeling was specifically bound when the antibodies were preincubated with protein A. After pronase digestion, both thyroglobulin-like material displayed different carbohydrate structures as judged by concanavalin A-Sepharose analysis. Thus, substituting multiantennary complex carbohydrate chains to the usual biantennary and high mannose structures present on thyroid-derived thyroglobulin had a profound effect on the prohormone immunoreactivity.

Porcine thyroid cells were cultured with or without thyrotropin for 9 days. It is known that the hormone enhances the synthesis of thyroglobulin, with an increase in the content and stability of its m-RNA. We show in the present work that thyrotropin also stimulates a number of glycosyltransferases diversely situated along the process of N-glycosylation. The most increased was oligosaccharyltransferase, responsible for attachment to nascent peptides of preformed core carbohydrate sequences. The relatively low activity level of oligosaccharyltransferase and its preferential responsiveness to thyrotropin supports the possibility of a regulation point at this enzyme.

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (125I-C/M ratio) decreases vs. days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 microU/ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 microU/ml thyrotropin; highest concentrations (4,000--16,000 muU/ml) are significantly inhibitory. In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d. Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34.3 pg/cell/d. The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties. Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.

An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization, lose their polarity and exhibit a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized the apical cell pole. In monolayer cells, on the other hand, the enzyme is distributed over the whole surface facing the medium. In both types of cultures fluorescence is also observed in intracytoplasmic organelles. In vivo, aminopeptidase ia a marker of the apical part of the thyroid plasma membrane, but its in vitro localization depends upon cell differentiation related to the culture conditions.

The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study revealed no change in the long-term export of thyroglobulin.

The OVNIS 6H and 5H thyroid cells, 2 permanent cell lines isolated 3 years ago from ovine tissue, synthesize a high molecular weight glycosylated protein, immunologically related to ovine thyroglobulin, which is similar to the prothyroid hormone dimer (17-19) S: thyroglobulin. Using sucrose gradient centrifugation and cell labelling with [14C]Leu or [3H]GlNH2, radioactivity was observed in proteins purified from cell layers and from cell culture media. Addition of thyrotropin to or removal from the media resulted respectively in an increase (+773%) or decrease (-1090%) of the total radioactivity detected in the (17-19)S thyroglobulin fraction. Estimation of thyroglobulin by RIA gave similar though less pronounced effects. These experiments prove (1) that thyroglobulin is still expressed in these OVNIS thyroid cell lines even after 3 years of permanent culture, (2) that TSH modulates the level of this protein through a TSH-receptor functional system.

The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4 degrees C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37 degrees C for 5-180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabeled. Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.

Porcine thyroid and embryo skin cells were isolated and cultured. Cultures from each kind of cells, as well as cocultures were fixed twice a week for electron microscopy. On day 8th a basal lamina like material appears only in the cocultures, along the basal plasmalemma of the reassociated thyroid cells. This amorphous material increases up to day 17th.

An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization have lost their polarity and exhibited a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized on the apical cell pole as observed in tissue. In monolayer cells, the enzyme is localized on all the surface facing the medium. In both types of cultures, fluorescence is also observed in intracytoplasmic organelles, probably lysosomes. Aminopeptidase is a marker of the apical part of the thyroid plasma membrane. Its in vitro localization depends upon cell differentiation which is in relation to culture conditions.

Thyrotropin given to support-anchored cultures of porcine thyroid cells, either in a serum-containing or in a serum-free system, produced an increase of about 50% in the total activity of 5'-nucleotidase. In the serum-free culture, in which TSH was administered to well-reformed follicles, this increase in 5'-nucleotidase activity concerns both the ecto-enzymic and intracellular forms of the enzyme and it coincides with the period of several days during which several glycosyltransferase activities are elevated and thyroglobulin production increased. Taken together, and in view of a recent in vitro study (Brandan and Fleisher, 1982) documenting the fate of uridine diphosphate in Golgi vesicles, these results suggest that there might be a functional correlation between the stimulation of 5'-nucleotidase and an increased production of nucleoside mono- and diphosphates when the activity of a number of glycosyltransferases is increased.

Thyroid cells in culture constitute a suitable system for the study of thyroid gland function and its regulation. The natural thyroid stimulator (TSH) induces the in vitro reorganization of thyroid cells into three-dimensional follicles morphologically and metabolically similar to gland follicles. In contrast, nonstimulated cells develop as a monolayer and in a concerted manner rapidly lose the enzymes involved in iodine metabolosm and the aptitude to bind TSH to specific receptor sites. The morphogenetic action of TSH and its ability to maintain the specific metabolic properties of thyroid cells in culture are mediated by cyclic AMP via new RNA and new protein synthesis. Therefore comparison of the properties of a given cell type in morphologically and metabolically different status should provide a valuable tool for studying the TSH mechanism of action. Using 125-I-labeled TSH of high purity, high specific radioactivity, and preserved biologic potency, TSH interaction with intact cells and their derived plasma membranes was studied. At both the cellular and the sub-cellular level a very good agreement was found for the values of the rate and equilibrium constants of labeled TSH binding. A single type of high-affinity low-capacity site was revealed. In contrast, in both systems dissociation of bound labeled TSH was not of single-order kinetics and showed two kinetic components with half-lives of 3 and 30 min. An excellent correlation between half-stimulation of adenylate cyclase and iodide transport mechanism activation, and the dissociation constant of TSH binding, was found, indicating that the in vitro system studied was relevant to physiologic regulation.

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.

Porcine thyroid cells in primary cultures form either monolayers or, when they are stimulated by thyrotropin (TSH), follicles. This system, monolayer-follicle associated cell culture, determines two morphofunctional states of the thyroid cell in vitro. These cells divide, when grown in monolayer. In this article we describe the precise conditions which allow the conversion of monolayer cells into follicles. The rate of cell growth was lowered using a serum-free medium. Cells were concentrated, stimulated by TSH and cultured on poly-L-lysine pretreated plastic flasks. Light and electron microscope studies show that cells reorganize into follicles containing thyroglobulin. Active iodide transport by the cells, as well as detection of thyroid hormones in the cell culture media, demonstrate that these follicles are functional. Formation of monolayers from follicles is in vitro a spontaneous phenomenon. It is linked to the loss of cell polarity, iodide transport, synthesis of hormones and to the decrease of the number of TSH receptor-sites. These main characteristics of differentiation may be regained in vitro after conversion of monolayer thyroid cells into active follicles up to at least generation five.

Primary cultures were initiated using thyroid tissue obtained at operation from patients with Graves's disease. The in-vitro conditions which permitted the formation of functional follicular structures in both primary cultures and derived sub-cultures were examined. In both situations, culture without the addition of calf serum to the medium resulted in the formation of follicles in response to thyrotrophin. In primary cultures the response to stimulation by exogenous thyrotrophin was variable. However, cells derived from long-term primary monolayers responded to thyrotrophin stimulation in a more predictable manner. In sub-cultures, the ability of cells to concentrate and organify iodide was augmented in a dose-dependent fashion in response to thyrotrophin (0 to 0.2 mu./ml); maximal values of 20 to 80 times those of control cultures being obtained. While follicular structure was maintained at higher hormone concentrations iodide-trapping capacity declined. Similar effects were produced by both low and high purity thyrotrophin and by dibutyryl cyclic AMP. Thyroid cells from two patients with a genetic defect of iodide organification exhibited the same lesion in vitro.

The hormonal regulation of thyroglobulin production has been studied using a clone of the ovine thyroid cell line: OVNIS 6H. 3 among the 6 hormones proposed for serum replacement are required for an optimal thyroglobulin production; insulin, hydrocortisone and thyrotropin. Insulin alone stimulates thyroglobulin production. The presence of insulin is also required to observe hydrocortisone and TSH stimulations. Newborn calf serum inhibits thyroglobulin production. The best conditions for optimal thyroglobulin expression and TSH responsiveness are obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.