Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.

In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation.

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity.

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.

Enhanced expression of the protein kinase substrate p36 in human hepatocellular carcinoma.

A basic phosphoprotein defined by a monoclonal antibody named AF5 was found to be highly abundant in human hepatocellular carcinoma by Western immunoblotting. Under the same conditions, the levels of this phosphoprotein were low or undetectable in normal liver extracts. The AF5 antibody was used to screen a cDNA expression library of a human hepatoma cell line named FOCUS. A 960-base-pair cDNA was isolated and found to be a partial cDNA encoding the human protein-tyrosine kinase substrate p36, also known as lipocortin II. p36 expression was highly abundant in hepatocellular carcinomas at both the transcript and protein levels. Its expression was not induced significantly during rat liver regeneration following a partial hepatectomy. These results suggest that the induction of p36 expression is associated with malignant transformation of hepatocytes. p36 was previously shown to be phosphorylated upon transformation of normal fibroblasts by retroviral oncogenes without significant modulation of expression. We report here the initial description of the association of increased p36 expression with malignant transformation.

Crystal structures of cellular retinoic acid binding proteins I and II in complex with all-trans-retinoic acid and a synthetic retinoid.

BACKGROUND: Retinoic acid (RA) plays a fundamental role in diverse cellular activities. Cellular RA binding proteins (CRABPs) are thought to act by modulating the amount of RA available to nuclear RA receptors. CRABPs and cellular retinol-binding proteins (CRBPs) share a unique fold of two orthogonal beta-sheets that encapsulate their ligands. It has been suggested that a trio of residues are the prime determinants defining the high specificity of CRBPs and CRABPs for their physiological ligands. RESULTS: Bovine/murine CRABP I and human CRABP II have been crystallized in complex with their natural ligand, all-trans-RA. Human CRABP II has also been crystallized in complex with a synthetic retinoid, 'compound 19'. Their structures have been determined and refined at resolutions of 2.9 A, 1.8 A and 2.2 A, respectively. CONCLUSIONS: The retinoid-binding site in CRABPs differs significantly from that observed in CRBP. Structural changes in three juxtaposed areas of the protein create a new, displaced binding site for RA. The carboxylate of the ligand interacts with the expected trio of residues (Arg132, Tyr134 and Arg111; CRABP II numbering). The RA ligand is almost flat with the beta-ionone ring showing a significant deviation (-33 degrees) from a cis conformation relative to the isoprene tail. The edge atoms of the beta-ionone ring are accessible to solvent in a suitable orientation for presentation to metabolizing enzymes. The bulkier synthetic retinoid causes small conformational changes in the protein structure.

Characterization of a malignant phenotype-associated cell surface glycoprotein common to various human tumor cells and preferentially expressed on adenocarcinoma of the lung.

Since both the liver and lung are derived from the endoderm, common antigens may appear on both tissues during malignant transformation. In an attempt to delineate cell surface alterations associated with the neoplastic transformation of these tissues, we have produced a library of monoclonal antibodies against a human hepatoma cell line termed FOCUS. One of these monoclonal antibodies, designated AF-10, recognized an antigen preferentially expressed on human lung adenocarcinoma cells, both in vitro and in vivo. This antigen has been characterized using Western immunoblot analysis and immunoprecipitation from surface-iodinated or metabolically labeled cells. The mature antigen is a cell surface glycoprotein with a core polypeptide with a molecular weight of 75,000 bearing N-glycosylation units. This protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 100,000-115,000 in reducing conditions and Mr 115,000-130,000 in nonreducing conditions. The epitope recognized by monoclonal antibody AF-10 is borne by the core protein. This antigen is shed from the cell surface and was identified in the culture supernatant from lung adenocarcinoma cell lines. Studies of the biodistribution of the AF-10 antigen showed that it was also expressed at low levels in normal human small intestine and kidney. The AF-10 monoclonal antibody may be useful for the study of the antigen expression between normal lung and the transformed phenotype.

In vivo localization of human colon adenocarcinoma by monoclonal antibody binding to a highly expressed cell surface antigen.

We have produced a monoclonal antibody (SF-25) against a human hepatoma cell line (FOCUS) that strongly reacts with an antigen shared by all six colon adenocarcinoma cell lines. This cell surface antigen was uniformly expressed in all 17 human adenocarcinomas of the colon obtained at surgery but not on the normal adjacent mucosa counterpart. Other normal tissues were negative except for a population of cells in the distal tubule of the kidney as shown by immunoperoxidase staining and direct binding to membrane preparations. Binding of this Mr 125,000 antigen to antibody is disrupted by detergents, sodium dodecyl sulfate, and paraformaldehyde fixation but not by treatment of FOCUS cells with trypsin. The SF-25 antibody when labeled with 125I shows a striking capacity by both biodistribution and nuclear imaging studies to localize human colon adenocarcinoma grown as solid tumors in nude mice. SF-25 may be useful in distinguishing between normal colon and the transformed phenotype.

Identification and characterization of a Mr 50,000 adrenal protein in human hepatocellular carcinoma.

A Mr 50,000 cell surface protein antigen (p50) was identified on a human hepatocellular carcinoma derived cell line (FOCUS) by two monoclonal antibodies (SF 31 and SF 90). This antigen was subsequently shown to be expressed in vivo in human hepatocellular carcinoma. All 18 tumors tested by Western immunoblotting demonstrated high levels of p50 with undetectable amounts observed in the adjacent normal liver counterparts. Further characterization revealed that p50 is a monomeric polypeptide with a neutral pI (6.5-7.2) and appears not to be glycosylated. The cellular localization was determined by direct antibody binding to intact cells, immunoprecipitation of 125I-labeled cell surface proteins, and Western immunoblotting of subcellular fractions. p50 was found on the cell surface as well as in the cytoplasm. In vitro monoclonal antibody binding studies indicate that the protein is expressed in all human malignant cells (n = 34) tested thus far regardless of the embryonic tissue of origin and the degree of differentiation. p50 was present at very low levels in normal tissues with the notable exception of high expression in adrenal glands. The protein is conserved in mammalian evolution since a similar protein was also found in bovine adrenals. The molecular characteristics and the pattern of expression of p50 indicate that this normal adrenal protein is associated with the transformed phenotype.

Identification and measurement of calcitonin precursors in serum of patients with malignant diseases.

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

Positive anticalcitonin immunoscintigraphy in patients with medullary thyroid carcinoma.

A cocktail of three monoclonal F(ab')2 fragments against three distinct epitopes of calcitonin or PDN 21 was labelled with either 111In or 131I. These F(ab')2 fragments, a control 125I-F(ab')2 fragment and 99mTc-pertechnetate were injected into four patients suffering from medullary thyroid carcinoma. Scintigraphy data were processed by energy factor analysis for an optimal separation of images corresponding to each isotope. The best tumor detection was obtained 1-3 days after injection of the 111In-F(ab')2 cocktail which clearly labeled the thyroid tumors in the four patients (smallest tumor detected, 0.6 cm) as well as lymph node and bone metastases. In the liver, positive detection was only successful with the 131I-labeled cocktail. These results were confirmed by counting rates of resected specimens which provided average specificity indices ranging from 3.3 to 13.1. Anticalcitonin antibodies could be particularly useful for immunoscintigraphy detection of residual or recurrent medullary thyroid carcinoma in patients with elevated calcitonin serum level.

Dopamine activates noradrenergic receptors in the preoptic area.

Dopamine (DA) facilitates male sexual behavior and modulates aromatase activity in the quail preoptic area (POA). Aromatase neurons in the POA receive dopaminergic inputs, but the anatomical substrate that mediates the behavioral and endocrine effects of DA is poorly understood. Intracellular recordings showed that 100 microm DA hyperpolarizes most neurons in the medial preoptic nucleus (80%) by a direct effect, but depolarizes a few others (10%). DA-induced hyperpolarizations were not blocked by D1 or D2 antagonists (SCH-23390 and sulpiride). Extracellular recordings confirmed that DA inhibits the firing of most cells (52%) but excites a few others (24%). These effects also were not affected by DA antagonists (SCH-23390 and sulpiride) but were blocked by alpha2-(yohimbine) and alpha1-(prazosin) noradrenergic receptor antagonists, respectively. Two dopamine-beta-hydroxylase (DBH) inhibitors (cysteine and fusaric acid) did not block the DA-induced effects, indicating that DA is not converted into norepinephrine (NE) to produce its effects. The pK(B) of yohimbine for the receptor involved in the DA- and NE-induced inhibitions was similar, indicating that the two monoamines interact with the same receptor. Together, these results demonstrate that the effects of DA in the POA are mediated mostly by the activation of alpha2 (inhibition) and alpha1 (excitation) adrenoreceptors. This may explain why DA affects the expression of male sexual behavior through its action in the POA, which contains high densities of alpha2-noradrenergic but limited amounts of DA receptors. This study thus clearly demonstrates the existence of a cross talk within CNS catecholaminergic systems between a neurotransmitter and heterologous receptors.

Production and in vitro utilization of monoclonal antibodies to human thyroglobulin.

Human thyroglobulin (Tg) was used as an antigen in the development of antibodies by the hybridoma technique. From four antibodies that bound more than 40% labeled Tg, two were characterized (182/E4 and 211/A5). They were both of the immunoglobulin G 2ab subclass, and provided an affinity constants (Ka) of 1.2 X 10(10) and 7.7 X 10(9) mol-1, respectively. The specificity of these antibodies was demonstrated by the absence of cross-reaction by monoiodothyronine, diiodothyronine, T3, T4, and sialic acid. A RIA was developed with 182/E4 or 211/A5, and the least detectable dose, based on the standard curve, was 10 ng/ml. The immunoreactivities of 182/E4 and 211/A5 to four Tg preparations different in iodine content appeared to be identical. Histochemical staining was used on normal and neoplastic tissues with both antibodies. Positive reactions were obtained in both cells and colloid, with heterogeneous staining from one follicle to another. Papillary carcinoma showed numerous positive cells, in contrast with Hürtle cell tumors which displayed very few positive cells. Anaplasic giant and spindle cells were negative. Monoclonal antibodies to human Tg are useful for in vitro detection of Tg.

Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis.

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

Radioiodination of human calcitonin using the Iodogen reagent.

A method is described for the radioiodination of human calcitonin using the Iodogen reagent. The tracer was purified by 2 successive gel filtrations. The first, using Sephadex G-10, resulted in the complete removal of free 125iodide as assessed by ascending paper chromatography. The second, involving Sephadex G-75, permitted the separation of the tracer from high molecular weight aggregates. Analytical RP-HPLC showed a slight proportion (13%) of a contaminating product, probably (sulfoxyl-methionine9)calcitonin. The final yield, after radioiodination, was found to be 42.7 +/- 5.5% and the tracer displayed a specific activity of 850 +/- 110 Ci/mmol. The effects of long-term storage of the frozen tracer were studied.

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label.

Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies.

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.

[Pruriginous dermatitis due to Pyemotes zwoelferi Krczal, 1963. About several cases in the Alpes-Maritimes (France) (author's transl)]. / Dermatite prurigineuse due à Pyemotes zwoelferi Krczal, 1963. a propos de plusieurs cas dans les alpes-maritimes.

In September 1978, people having had contacts in the neighbouring of Nice with an everlasting flower package (Helichrysium angustifolium) from Jugoslavia, complained of prurigo. The erythematous lesions with a central vesicle regressed during the second week but did not fade out until the third week. Among the everlasting flowers were found numerous and swiftly mowing fasting females of Pyemotes zwoelferi. Those mites are usually parasitic on phytopathogenic insects. Their aggressiveness against human beings is related to sudden development of their population and, also to the specific toxicity of their venom. Systematically P. zwoelferi comes in the same species group as P. tritici.

Benzodiazepine receptors on human blood platelets.

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.

[Epidemic aspect of parasite pruriginous dermatitis of Pyemotes zwoelferi type (author's transl)]. / Une épidémie de dermatite prurigineuse parasitaire à Pyemotes zwoelferi.

The responsibility of the Pyemotes in the genesis of certain parasite prurigo is known since the middle of the 19th century. The epidemic that we are describing owes its originality to the species Pyemotes zwoelferi sent in a bunch of dried flowers coming from Yugoslavia. The affection of man by these Pyemotes is essentially due to two factors: one of them is of an ecological nature due to the violent change of the conditions under which the acarian population grew and developed, and the other one linked to the species and the belonging to the ventricosus group. The varieties of the ventricosus group have a host specificity which is by far less narrow than that of the scolity group and they are also the most pathogenic to man.