Interaction of local anaesthetics with cytochrome oxidase studied with fluorescence quenching.

The interaction of a series of eight local anaesthetics with cytochrome oxidase chosen as a membrane model protein has been studied with fluorescence technique using quinacrine as a fluorescent probe. The existence of hydrophobic interactions with a non polar region of cytochrome oxidase complex has been shown. The ability of the drug molecules to displace quinacrine bound to cytochrome oxidase correlate as closely with their anaesthetic potency as with their octanol-water partition coefficient. Our results are in good agreement with a recent model of local anaesthetic action on nerve membranes presenting a site of anaesthesia including both lipid binding and protein binding environments.

Inhibition of cytochrome oxidase activity by local anaesthetics.

Using a polarographic method, we studied the inhibition of mitochondrial electron transport at the cytochrome c oxidase site caused by eight local anaesthetics. The diversity of the types of inhibition observed indicate the importance of electrostatic interactions between the anaesthetic molecules and the membrane protein. A linear relationship was recognized between the anaesthetic activity of infiltration and the affinity for the enzyme. We also observed a significant relationship between this affinity and the octanol-water partition coefficient. This result suggests that lipophilic interactions are involved in cytochrome oxidase-anaesthetic binding. We tried to establish a parallel between this binding and the mechanism of anaesthesia involving the nerve membrane proteins.

Evidence that nicotine acetylcholine receptors are not the main targets of cotinine toxicity.

The toxicity of nicotine, cotinine and their mixtures was studied in Mus musculus mice as well their effects on growth after repetitive administration to young mice. The affinity constants of the two alkaloids for the nicotinic acetylcholine receptors (nAChRs) of Torpedo and rat brain membranes were determined. The administration of these alkaloids produced distinct symptoms of intoxication. Nicotine was 100-fold more toxic than cotinine and 10-fold more rapid than cotinine at producing respiratory arrest. The affinity of nicotine for both subtypes of nAChRs was > 100-fold higher than that of cotinine. Repetitive administrations of nicotine caused weight loss, whereas that of cotinine caused weight gain (P < 0.01). The administration of the two alkaloids as mixtures to mice caused significantly (P < 0.01) higher mortality than theoretically expected. Furthermore, hexamethonium pretreatment reduced by 2-fold (P < 0.01) the toxicity of nicotine but enhanced by 1.6-fold (P < 0.01) that of cotinine and was without effects on toxicity of mixtures. We suggest that nAChRs are not the main targets of cotinine toxicity.

Expression of estrogen receptors during growth of human pancreatic adenocarcinoma cells (Capan-1)-relationship with differentiation.

In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic duct cells, the expression of estrogen receptor (ERalpha) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1 cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERalpha and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERalpha (236 +/ 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%). At the onset of the stationary phase (Day 5), ERalpha levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 +/- 0.9 fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen. Immunocytochemical analysis with anti-ERalpha-1D5 antibodies showed nuclear and cytoplasmic localization of ERalpha in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERalpha is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in nontarget tissues.

[Long-term prognostic role of steroid receptors in cancer of the breast]. / Rôle pronostique à long terme des récepteurs stéroïdiens dans le cancer du sein.

We screened for the prognostic value of estrogen receptor (ER) and progesterone receptor (PR) through a multicentric study of 2,257 operable breast cancer patients who did not received adjuvant therapy. Three hundred and seven local-regional recurrences, 105 metachronous contralateral breast cancer, 589 metastases and 537 deaths from cancer had been diagnosed with a median follow-up of 8.5 years. A total of 69% of the tumors were ER positive and 54% PR positive. For statistical analysis, 1,665 patients were studied because of complete clinical and biological data. In univariate analysis, ER and PR status were of prognostic value for the metastases-free interval (MFI) and the overall survival (OS). In multivariate analysis (Cox proportional hazard model), only the ER status showed a significant difference between positive and negative groups regarding the MFI and OS. By using Cox regression model with time-dependent covariates, we show that the predictive value of ER status of the primary tumor decreases by approximately 20% per year, losing its significance after 8 years of follow-up. These results show that ER and PR status have a relatively limited predictive value and their major interest remain in the domain of therapeutic decision.

Heterotransplantation of a human oligoastrocytoma into nude mice: difference in tumour growth between males and females.

A human oligodendroglial tumour cell line was established in the nude mouse from a mixed oligoastrocytoma. This cell line was identified by immunoperoxidase screening for glial fibrillary acid protein (GFAP) and Leu-7, by electron microscopy, by its growth behaviour, and by radioreceptor assays for oestrogen, progestogen and androgen. At the first heterotransplantation, the tumour grew in the female but not in the male mice. GFAP-positive cells were much less abundant than in the initial tumour, although Leu-7 positive cells were plentiful. Growth curves were biphasic, and the tumours contained both oestrogen and androgen receptors. After three successive heterotransplantations in female mice, GFAP-negative, Leu-7-positive tumours with the cytological features of an oligodendroglioma were obtained. In contrast to those of the first heterotransplantation, the growth curves showed a longer latency and a single growth phase. The tumours contained both progestogen and androgen receptors but lacked oestrogen receptor. The 5th heterotransplantation in male mice exhibited rapid growth, similar to the three previous heterotransplantations in the female mice. The growth observed solely in the female mice at the first heterotransplantation was thought to derive from the hormonal status of the host. The presence of hormone receptors in the tumours at the first heterotransplantation lent support to this idea. There was no further influence of the sex of the host on tumour growth once the oligodendroglial line was established. The establishment and the development of oligodendrocytes may result from differentiation of transitory cells intermediate between astrocytes and oligodendrocytes. The effect of host-sex on the development of oligodendrocytes points to a new facet of the differentiation of CNS glial cells.

Influence of host sex on the growth of a human glioblastoma line in athymic mice.

Glioblastomas are generally thought to be more common in men than in women. In order to investigate the hormone-dependence of these tumours, we established a human glioblastoma line in athymic mice. The tumour cell type was characterized using immunocytochemical methods. The influence of host sex on growth was evaluated, and hormone receptors were characterized biochemically. The histological features of the initial tumour were conserved in the hetero-transplanted tumours, which consisted of vimentin and GFAP immunoreactive astrocytes. There was a highly significant difference in tumour growth between the two sexes (P less than 0.01). In the male mice, tumours were from 2.5 to 10 times larger than in the females, the latency periods were 30% shorter, and the growth phases were characterized by periods of slow or zero growth. In addition, androgen and oestrogen receptors were detected at low levels (80-270 fmol/g tumour) in the heterotransplanted tumours especially in the males. The fact that the male tumour growth profiles resembled those of some hormone-dependent lines, and that androgen receptors were found preferentially in the male rather than in female tumours would tend to indicate that there is a hormonal influence on the growth of the heterotransplanted tumours. These results provide further evidence for an influence of sex-steroid hormones on the growth of glioblastomas.

Hormonal steroid receptors in intracranial tumours and their relevance in hormone therapy.

Determination of cytosolic estradiol and progesterone receptors was carried out in 30 intracranial tumors: 12 meningiomas, 13 metastases, two angioreticulomas, two gliomas and one sarcoma. The hormonal fraction found with the receptor and the dissociation constant (Kd) were determined by the Scatchard method. Values higher than 10 fmol/mg of protein were considered as positive. Ten of the 12 meningiomas (83%) showed progesterone receptors (RP), while estrogen receptors (RE) were not found in any of the cases; six of the 13 metastases showed the two types of receptors; the other tumors did not present detectable receptor levels. There was no correlation between the receptor level and patient age, sex or hormonal status. Thee results suggest the possible use of endocrine therapy for example in cases of high-risk patients or incomplete surgical resection.

[Conformation properties and pharmacological activity of adrenergic catecholamines]. / Propriétés conformationnelles et activité pharmacologique de catécholamines adrénergiques.

In this study, the authors propose a comparison between conformational properties and pharmacological activity of a series of adrenergic phenethylamines; in particular they conclude that if the alpha adrenergic compounds adopt a gauche conformation, beta adrenergics prefer a trans form.

[Variation in the number of estrogen and progesterone receptors in normal human endometrial cells in culture]. / Variation du taux récepteurs des oestrogènes et de la progestérone dans les cellules endométriales humaines normales en culture.

As estrogens are in vivo potent growth factors of their target cells but are ineffective in vitro on human endometrial cell multiplication, an eventual role of steroïd sex hormone receptors has been suspected. These receptors have been assayed in human endometrium, and cytoplasmic concentrations compared between fresh tissue, short term and long term cultures. Receptors remain present in culture but at a lower level than in vivo. Estrogen receptors keep stable with the duration of the culture while progesterone receptors progressively diminish. More, these receptors are active: estradiol administration induces a new progesterone receptor synthesis in culture. So, steroïd sex hormone receptors do not seem to be involved in the ineffectiveness of estrogens on cell multiplication in culture.

Isolation and microsequencing of a novel cotinine receptor.

1. Nicotine and its main derivative. cotinine, are reported to have distinct central activities in mammals. In this study, the cotinine receptor was separated by biochemical procedures including radio receptor, affinity-chromatography, SDS-PAGE, and N-terminal sequencing assays. 2. Consistently, the results showed that distinctive cotinine receptors exist in different tissues of mammals. In rat brain, the affinity chromatography and [125I]cotinine receptor essays were used to isolate a 40-kDa protein (p40) with higher affinity for cotinine than alpha-bungarotoxin and nicotine. The N-terminus amino acid sequences of the p40 and its internal tryptic peptides showed no identity to recently described protein sequences, with the exception of homology to the human p205 synovial fluid protein. 3. These results, in agreement with other behavioral studies, are the first molecular evidence for distinctive nicotine and cotinine receptors in mammals.

Immunoradiometric assay of thyroglobulin in patients with differentiated thyroid carcinomas: need for thyroglobulin recovery tests.

As interference from thyroglobulin autoantibodies appears to have been overcome in new commercial thyroglobulin assays by the use of monoclonal antibodies, the need for thyroglobulin recovery tests became uncertain. Sera (n=45) from patients with differentiated thyroid carcinomas were selected on the basis of a thyroglobulin recovery value below 70% in the Dynotest Tg immunoradiometric assay (Brahms) routinely used in our laboratory. Serum thyroglobulin levels were then measured using three other commercial immunoradiometric assays: thyroglobulin ERIA (Pasteur), HTGK (Sorin) and ELSA HTG (Cis Bio International). Thyroglobulin autoantibodies were measured using the Thyrak assay (Brahms). Although many patients were thyroglobulin antibodies-negative (< 200 U/ml, n=26), most immunoradiometric assays failed to detect thyroglobulin in patients with evidence of recurrence. Low thyroglobulin values associated with low thyroglobulin recovery in thyroglobulin antibody-negative patients appear to be more biologically relevant than a single low thyroglobulin value, which can lead to lack of medical intervention. We conclude that the thyroglobulin recovery test is a prerequisite for the correct interpretation of serum thyroglobulin levels determined with immunoradiometric assays in the follow-up of thyroglobulin autoantibody-negative patients treated for differentiated thyroid carcinomas.

Nicotine is more efficient than cotinine at passing the blood-brain barrier in rats.

1. Nicotine and its main metabolite, cotinine, were reported to have distinct behavioral activities in mammals. 2. In this study, cotinine was synthesized without detectable nicotine contamination to compare the ability of nicotine and cotinine to pass the blood-brain barrier (BBB) in rats. 3. The alkaloids were extracted from plasma and brain tissues by methanol, identified by thin-layer chromatography, and quantified by high-pressure liquid chromatography and radioimmunoassays. 4. Consistently, the three methods showed that the passage of cotinine was time, route of administration, and dose dependent and that nicotine was more efficient than cotinine to pass the BBB. 5. The results suggest that these alkaloids may have central activities that probably result from their actions at distinct molecular levels.

Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers.

The prognostic value of oestrogen receptor (ER) and progesterone receptor (PR) was estimated through a multicentric study of 2257 operable breast cancer patients followed up for a median of 8.5 years. None of the patients had received adjuvant therapy. The series included 33.3% stage I patients, 57.1% stage II, 5.7% stage IIIa and 2.4% stage IIIb. At the end point of the study 589 metastases and 537 deaths from cancer were recorded. Receptor measurements were performed by radiolgand assay according to a uniform protocol. A total of 68.8% of the tumous were ER positive and 54.0% PR positive ( > or = 10 fmol mg-1 cytosol protein). In univariate analysis, ER and PR status (positive/negative) were of prognostic value (P < 0.001) for the disease-free interval (DFI), the metastases-free interval (MFI) and the overall survival (OS). The OS of the patients after a first metastasis was also significantly different between ER-positive and -negative tumours (P < 0.001). In multivariate analysis (Cox proportional hazard model, 1665 patients), only the ER status showed a significant difference (P < 0.01) between positive and negative groups regarding the DFI, MFI and OS. By using Cox non-proportional, time-dependent models, we show that the predictive value of ER status of the primary tumour decreases by approximately 20% per year, losing its significance after 8 years of follow-up. Overall, when compared with TNM and histological grading, ER and PR status have a low prognostic value, their major interest remaining solely in the domain of therapeutic decision.