Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Ferritin H gene polymorphism in idiopathic hemochromatosis

Summary We have analysed karyotypes and DNA from three patients with aniridia (congenital absence of irises) and Wilms' tumour. All three had constitutional deletions from the short arm of chromosome 11. The minimum region of overlap of the deletion involves a small region of band 11p13 presumed to contain the genetic loci responsible for both phenotypic abnormalities. Using cells from these patients, somatic cell hybrids with transformed mouse cells have been prepared. Individual subclones retaining either the deletion-11 chromosome or the normal chromosome 11, in addition to a variety of other human chromosomes, have been identified. The relative position of these breakpoints have been determined and the panel of hybrids has been used to map randomly-isolated 11p13 DNA sequences. The characterisation of these deletions has provided a useful panel of hybrids for random mapping strategies designed to identify the Wilms' and aniridia genes.     

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.     

Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.