Expression in transgenic mice of the large T antigen of polyomavirus induces Sertoli cell tumours and allows the establishment of differentiated cell lines.

The large T antigen of polyomavirus (PyLT) efficiently immortalizes rodent fibroblasts, but, unlike SV40 T antigen, it is not sufficient to achieve complete oncogenic transformation. We analysed a series of transgenic mouse families that express the PyLT protein under control of the viral enhancer-promoter region. In all of them, the transgene was expressed in the seminiferous epithelium of the testis (Sertoli and germ cells), with no pathological consequences during most of the animals' lives. However, every old male developed large bilateral tumours of the testes, generated by the proliferation of Sertoli cell derivatives. Cell lines could be readily established both from the tumours and from the still apparently normal testis before the onset of tumoral growth. They retained in vitro morphological and ultrastructural features characteristic of Sertoli cells. But, in addition to this major Sertoli component, the maintenance of a cellular contingent of germinal origin was suggested by the expression of genes that are normally transcribed during the premeiotic and early meiotic stages of spermatogenesis (LDH-X, Hox1.4 and c-kit). The two cell types remained tightly associated, even at late passages in culture, and could not be separated by conventional cloning procedures. This association in culture of the two cell types whose interaction is critical for spermatogenesis may provide a useful tool for its molecular analysis.

Thyrotropin induces growth and iodothyronine production in a human thyroid cell line without affecting adenosine 3',5'-monophosphate production.

Using a recently described cell line derived from the fusion of human thyroid and human myeloma cells, we studied the effects of TSH on cell growth, iodide organification, and cAMP production. Although these cells grow in the absence of TSH, when incubated for 2 days in serum-free medium containing purified human or bovine TSH, there was a significant increase in cellular DNA content. The stimulatory effect was observed at concentrations as low as 0.5 microU/ml, which produced a significant increase in DNA content, and was maximal with 5 microU/ml. This effect was still present after 6 days of incubation. Electron microscopy performed by an unbiased observer on cells incubated in the presence of TSH showed an increase in the calculated size of the cells and the nucleus which was significant at 0.1 microU/ml and maximal at 1 microU/ml. Stimulation of 125I organification and hormone production was observed at TSH concentrations as low as 1 microU/ml and was maximal at 10 microU/ml. However, neither TSH (1-50 microU/ml) nor forskolin (10(-6) M) stimulated cAMP production. These data suggest that these cells lack a functional adenylate cyclase pathway and that growth and iodide organification are mediated by other second messenger systems. Such a cell line could yield new insights into the mechanisms of TSH action and may provide a sensitive bioassay for TSH and other thyrotropic factors.

Distribution of exogenous 25-hydroxycholesterol in Hep G2 cells between two different pools.

Binding of [26,27-(3)H]25-hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse-chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t(1/2) of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27-(3)H]25HC in the presence of a 30-fold molar excess of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one was found to cause (i) 2-fold decrease in the binding of [26,27-(3)H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4-5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites.

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.

UNLABELLED: Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. IN CONCLUSION: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of hormone replacement therapy on age-related increase in carotid artery intima-media thickness in postmenopausal women.

The aim of this study was to determine the changes in carotid artery intima-media thickness as measured by B-mode ultrasound in postmenopausal women receiving hormone replacement therapy (HRT) or not. One hundred and fifty-nine healthy postmenopausal women aged 45-65 years were recruited from our menopause clinic. All the selected women were free of cardio-vascular diseases and had no cardio-vascular risk factors. None of the women were receiving lipid-lowering or antihypertensive drugs. Because carotid artery intima-media thickness was shown to be strongly and positively correlated with age in women aged 55 years and older but not before, women were divided into four groups according to age (<55 vs. > or =55 years) and use of HRT (current users vs. never users). All the treated women received non-oral 17beta-estradiol with a non-androgenic progestin and had started HRT within the first year after menopause. Scanning of the right common carotid artery was performed with a B-mode ultrasound imager and thickness of the intima-media complex as well as luminal diameter of the artery were determined using an automated computerized procedure. Within each age group (i.e. <55 or > or =55 years), women had comparable demographic characteristics and only differed by HRT use. Long-term treated women had significantly lower total cholesterol levels than untreated women (P=0.005). Triglycerides, low-density lipids (LDL)-cholesterol and high-density lipids (HDL)-cholesterol levels, systolic and diastolic blood pressure were not significantly different between users and non-users. In women <55 years, no significant difference in carotid intima-media thickness was found between current users (mean 2.5+/-1.4 years) and non users. In older women, the mean values of carotid intima-media thickness were significantly smaller in current users (mean 6.9+/-3.3 years) than in never treated women: 0.50+/-0.05 versus 0.56+/-0.07 mm, P<0.0001. Carotid artery intima-media thickness was significantly correlated to age in never users (r=0.5, P<0.0001) but not in women who were currently receiving HRT (r=0.2, ns). These findings suggest an apparent protective effect of long-term HRT on age-related thickening of the intima-media of the right common carotid artery. This may contribute to explain the apparent cardio-protective effect of HRT after the menopause.

Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase.

A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH.

Immunocytochemical study of localization and traffic of thyroid peroxidase/microsomal antigen.

We studied the distribution of binding sites for anti-peroxidase monoclonal antibody and anti-microsomal antibodies on isolated human thyroid follicles and a human thyroid cell line. Both open follicles and cells were incubated first with antibodies at +4 degrees C, then with colloidal gold labelled protein A. The topography of the binding sites for monoclonal anti-peroxidase antibody corresponded closely to the expected cell surface distribution of endogenous thyroid peroxidase since labelling was observed at the apical cell surface of the follicles. Furthermore, labelling was restricted to the microvilli level; while smooth membrane territories were devoid of binding sites. In some cases, incubations at 4 degrees C were followed by warming the follicles and cells up to 37 degrees C for 20 minutes in order to study internalization of ligands. Ligands were then observed in intracellular organelles: endosomes and lysosomes. Essentially the same results were observed when human antibodies to the microsomal antigen were used. Controls with microsomal antibodies depleted in anti-peroxidase were negative. In conclusion these findings show that: 1) thyroid peroxidase is present in limited areas on the apical cell surface, 2) labelling of follicles and cells by the anti-microsomal antibodies had the same pattern of distribution as the monoclonal anti-peroxidase antibody, thus suggesting that they recognize the same apical antigens, and 3) TPO/MIC antigen traffics from the cell surface towards lysosomes when the cells are incubated at 37 degrees C.

A double blind parallel group placebo controlled comparison of sedative and mnesic effects of etifoxine and lorazepam in healthy subjects [corrected].

This paper describes the psychomotor and mnesic effects of single oral doses of etifoxine (50 and 100 mg) and lorazepam (2 mg) in healthy subjects. Forty-eight healthy subjects were included in this randomized double blind, placebo controlled parallel group study [corrected]. The effects of drugs were assessed by using a battery of subjective and objective tests that explored mood and vigilance (Visual Analog Scale), attention (Barrage test), psychomotor performance (Choice Reaction Time) and memory (digit span, immediate and delayed free recall of a word list). Whereas vigilance, psychomotor performance and free recall were significantly impaired by lorazepam, neither dosage of etifoxine (50 and 100 mg) produced such effects. These results suggest that 50 and 100 mg single dose of etifoxine do not induce amnesia and sedation as compared to lorazepam.

[Steryl cellosolves--regulators of cholesterol metabolism in isolated rabbit hepatocytes]. / Steriltsellozoly'vyv--reguliatory metabolizma kholesterina v izolirovannykh gepatotsitakh krolika.

Synthesis of 3 beta-(2-hydroxyethoxy)cholest-5-ene, 3 beta-(2-hydroxyethoxy)cholest-5-en-7-one, 3 beta-(2-hydroxyethoxy)-7 beta-hydroxycholest-5-ene, 3 beta-(2-hydroxyethoxy)-5 alpha, 6 alpha-epoxycholestane, and 3 beta-(2-hydroxyethoxy) -5 alpha, 6 beta-dihydroxycholestane was described. Substances obtained inhibited cholesterol biosynthesis in the rabbit hepatocyte cell culture with ID 50 from 5.5(+/-0.7) x 10(-8) to 1.3(+/-0.2) x 10(-5) M and also to a remarkable extent the cell protein biosynthesis.

[Metabolism of 3beta-(2-hydroxyethoxy)cholest-5-ene after intravenous administration to rats]. / Metabolizm 3beta-(2-gidroksiétoksi)kholest-5-ena pri vnutrivennom vvedenii krysam.

The intravenous injection of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest- 5-ene into rats with the cannulated common bile duct resulted in the primary accumulation of radioactivity in liver (24%) and spleen (12%) tissues, bile (7%), and blood serum (15%) after 3 hours. The distribution of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene throughout various tissues was close to that of [14C]cholesterol being administrated under the same conditions. The analysis of radioactive products from blood serum showed that 40-60% of 3 beta-(2-hydroxy-2-[3H]ethoxy)- cholest-5-ene was converted to the acyl derivative under experimental conditions.

[Affinity of 3-beta-(2-hydroxyethoxy)-5-alpha-cholest-8(14)-ene-15-one to oxysterol binding protein and its metabolism in HepG2 hepatoma cells]. / Srodstvo 3beta-(2-gidroksiétoksi)-5alpha-kholest-8(14)-en-15-ona k oksisterinsviazyvaiushchemu belku i ego metabolizm v kletkakh gepatomy HepG2.

beta-(2-Hydroxyethoxy)-5 alpha-cholest-8(14)-en-15-one, a synthetic inhibitor of cholesterol biosynthesis, was shown to exhibit a high affinity to oxysterol binding protein. This was proved by ultracentrifugation of the protein fraction from rabbit liver in the presence of the 3H-labeled inhibitor, 3 beta-(2-hydroxy-2-[3H]ethoxy)-5 alpha-cholest-8(14)-en-15-one, or by the substitution of the [3H]-25-hydroxycholesterol in its complex with the oxysterol binding protein. In human hepatoma Hep G2 cells, the inhibitor decreased activity of 3-hydroxy-3-methylglutaryl CoA reductase [ID50 (2.7 +/- 0.7) x 10(-5) M] and was transformed into 3 beta-[2-(9-Z-octadecenoyloxy)ethoxy]-5 alpha-cholest-8(14)-en-15-one.

[Cross-sectional study of the prevalence of adjustment disorder with anxiety in general practice]. / Etude transversale de la prévalence du trouble de l'adaptation avec anxiété en médecine générale.

Although Adjustment Disorder (AD) is considered a marginal diagnostic category by many clinicians and researchers, all the rare studies undertaken in the last decades indicate that the prevalence of this disorder is high in psychiatric settings, but has never been investigated in general practice. The purpose of this study was to evaluate the current prevalence of Adjustment Disorders With Anxiety (ADWA) in primary care settings and to describe the characteristics of the population, nature of the stressors and management of the disorder by General Practitioners (GPs). This French study involved 78 random liberal GPs, in 7 distinct regions (Paris, Lille, Bordeaux, Rouen, Dijon, Castres and Compiègne). GPs had to register all the consecutive attenders over 18 years old. For each physician, the registration period was over when 200 patients were registered, or 10 days of consultation were completed, or when 5 MINI had been performed. The average study period was 10 days per physician. At the first stage, they selected all the patients with psychological complaints, which were eventually associated to physical complaints. At the second stage, only the patients whose complaints were linked to a psychosocial stressor and without A1 and/or A2 DSM IV criteria for a Major Depressive Episode (MDE) were proposed the Mini International Neuropsychiatric Interview (MINI). The MINI is a brief structured clinical diagnostic interview that identifies the main axis-I DSM IV diagnoses in about 15 minutes. Before starting the study, all of the GPs participated in an intensive course on AD criteria recognition and were trained to use the MINI. The GPs registered a total of 7,759 consecutive patients. Twenty-two per cent (n = 1,719) of the patients reported psychological complaints, associated or not to physical complaints. Among them, 49% (n = 844) linked their complaints to identifiable psychosocial stressors. About half of the latter (n = 450) coded positive to A1 and/or A2 criteria for MDE. At the end, a total of 314 patients agreed to complete the MINI. Among the 1,719 patients with psychological complaints, the prevalence of ADWA eventually associated to other psychiatric disorders was 9.2%. The prevalence of "pure" ADWA was 4.5%. When considering the whole population of consecutive patients in primary care settings, the prevalence of pure ADWA was 1.0%. Patients suffering from pure ADWA were mostly women (66.7%), young patients (mean age: 42 years), with a professional activity. Patients had a psychiatric disorder history in 53.8% of the cases (mostly anxiety disorder). The main life events cited as being responsible for the disorder were work-associated problems (23.1%), followed by family illness (9.0%) and serious personal illness or accident (7.7%). The average duration of the disorder was 2.32 months. In 91% of the cases, GPs estimated that the patient required a pharmacological or psychological treatment. In most cases, they treated the patients with drug therapy (74.0%) associated with psychological support (counselling or psychotherapy, 76%). Anxiolytic agents were usually prescribed (64.9%), followed by antidepressants (10.8%) and hypnotics (8.1%). In conclusion, this first prevalence study of ADWA in general practice demonstrates that this disorder is frequent in primary care. It seems to be more present in patients who are of working age, especially women. ADWA would thus seem to preferentially affect active subjects. In most cases, GPs treat their patients with both psychological support and drug therapy. Anxiolytic is the elicited treatment of this disorder.

Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a double-blind controlled study in general practice.

Adjustment Disorders With Anxiety (ADWA) account for almost 10% of psychologically motivated consultations in primary care. The aim of this double-blind randomised parallel group study was to compare (non-inferiority test) the efficacies of etifoxine, a non-benzodiazepine anxiolytic drug, and lorazepam, a benzodiazepine, for ADWA outpatients followed by general practitioners. 191 outpatients (mean age: 43, female: 66%) were assigned to receive etifoxine (50 mg tid) or lorazepam (0.5-0.5-1 mg /day) for 28 days. Efficacy was evaluated on days 7 and 28 of the treatment. The main efficacy assessment criterion was the Hamilton Rating Scale for Anxiety score (HAM-A) on Day 28 adjusted to Day 0. The anxiolytic effect of etifoxine was found not inferior to that of lorazepam (HAM-A score decrease: 54.6% vs 52.3%, respectively, p=0.0006). The two drugs were equivalent on Day 28. However, more etifoxine recipients responded to the treatment (HAM-A score decreased by >or=50%, p=0.03). Clinical improvement (based on Clinical Global Impression scale CGI, Social Adjustment Scale Self-Report SAS-SR, and Sheehan scores) was observed in both treatment arms, but more etifoxine patients improved markedly (p=0.03) and had a marked therapeutic effect without side effects as assessed by CGI, p=0.04. Moreover, 1 week after stopping treatment, fewer patients taking etifoxine experienced a rebound of anxiety, compared to lorazepam (1 and 8, respectively, p=0.034).

The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role.

The binding characteristics of the GlcNAc binding protein present in thyroid membranes (Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J.E., Salvatore, G., and Kohn, L.D. (1981) J. Biol. Chem. 256, 10592-10599) were reinvestigated using neoglycoproteins as probes. Plasma membrane preparations from porcine thyroid specifically bound 125I-GlcNAc35-bovine serum albumin. Binding was dependent on the presence of calcium. Binding of ligand to receptor was minimal at neutral pH and maximal at pH 5.0. Equilibrium binding studies indicated positive cooperativity of binding and a site capacity of about 60 pmol/mg of protein. Competition studies were compatible with a specificity hierarchy of GlcNAc much greater than Gal; no recognition of mannose, fucose, or glucose moieties was noted. The receptor was detergent-solubilized from plasma membrane preparations and on the basis of the defined binding properties, purified by chromatography on a GlcNAc-Sepharose affinity column. The purified GlcNAc thyroid receptor has a subunit molecular size of about 45 kDa and appears to be an oligomer composed of three subunits. The receptor was identified as a component of thyrocytes by in situ cytochemical localization with fluorescent neoglycoproteins. In certain cases it was mainly present on, or near, the apical cell surface. It is suggested that this GlcNAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyroglobulin molecules back into the follicular lumen.

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.

Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland.

A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.

Expression of Ia antigens on freshly dissociated mouse thyroid follicles demonstrated by immunofluorescence.

Iak antigens were detected by indirect immunofluorescence on CBA mouse thyroid follicles. Isolated thyroid follicles, free of lymphocyte contamination, were obtained by collagenase treatment and mechanical disruption; they were then cytocentrifuged on glass slides. This material was incubated with polyclonal or monoclonal anti Iak antibodies followed by FITC-conjugated F(ab')2. Subsequent microscopic observations of these open thyroid follicles revealed that Iak antigens were spontaneously expressed. Labelling was distributed preferentially on the thyrocyte cell surface and, to a lesser extent, on intracellular organelles. Conversely, nuclei were never stained. In some cases, labelling was mottled. Ia antigens may have been previously unobserved because, beside autoimmune disorders they are present in such small amounts that detection is very difficult. The fact that Ia expression is independent of morphological signs of spontaneous autoimmune thyroiditis (particularly, lymphocyte infiltration of follicles), raises the problem of correlation between the autoimmune disorders and Ia expression.

[Demonstration of a basal lamina in cocultures of thyroid cells and embryonal swine skin cells]. / Mise en évidence d'une lame basale dans des cocultures de cellules thyroïdiennes et de cellules provenant de la peau de Porc.

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.

Biosynthesis of the basal lamina as a result of interaction between thyroid and mesenchymal cells in culture.

Porcine thyroid cells were cultured alone or in mixed cultures with mesenchymal cells. The formation of a basal lamina in vitro was investigated ultrastructurally. Follicular reassociation of thyroid cells occurred in both types of culture; however, it was followed by formation of the basal lamina only when mesenchymal cells were present. The present findings suggest an epithelial origin of the basal lamina resulting from an interaction with mesenchymal cells.

Interaction of tubulin with chromatin proteins. H1 and core histones.

Purified histones, H1 or core histones, induce the aggregation of tubulin. The aggregation process, studied by light scattering at 350 nm and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is dependent on the respective amounts of histones and tubulin; a maximum is obtained at a stoichiometry of 1 molecule of H1 or 2 molecules of core histones/tubulin dimer. At these molar ratios, all tubulin and histone molecules are found in the insoluble material which sedimented at 2000 X g. Increasing H1 or core histones, there is a progressive decrease of light scattering and a concomitant formation of soluble complexes. The minimum soluble complexes between tubulin and H1 or between tubulin and core histones have the same apparent molecular weight of 150,000-160,000; these complexes consist of one tubulin and two H1 molecules or one tubulin and four core histones. The tubulin-histone interaction is an almost instantaneous reaction which can, however, be slowed down by increasing the ionic strength of the medium. NaCl (0.5 M) decreased by 50% the formation of tubulin-H1 insoluble complexes but slightly affects the core histones-tubulin complex formation. Histones can be classified by their ability to form insoluble complexes with tubulin: H4 = H3 greater than H2B = H2A greater than H1. The reactivity of histones seems to be related to their lysine/arginine content. Electron microscopy revealed that insoluble polymers resulting from the interaction of tubulin with H1 or core histones are similar and consist of unordered aggregates of 35-40 nm ring structures.