Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Kerntrockengewicht und Beteiligung von 3H-Thymidin an der DNS-Synthese in Einzelzellen der regenerierenden Rattenleber

Zusammenfassung Autoradiographische und interferometrische Untersuchungen an Tupfpräparaten der regenerierenden Rattenleber ergeben eine nahezu quantitative Beziehung zwischen Kerntrockengewicht und Einbau von ³H-Thymidin in die DNS. Zur Bestimmung des relativen ³H-Thymidingehalts des Einzelkerns wurde die Silberkornzahl auflichtphotometrisch bestimmt und für die Koinzidenz der -Teilchen sowie für die -Selbstabsorption korrigiert. Die Ergebnisse lassen eine lineare Korrelation zwischen Kerntrockengewicht und ³H-Thymidineinbau zweifelhaft erscheinen.

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Summary Autoradiographic and interferometric investigations have been performed in single nuclei from the regenerating liver of the rat. The results indicate an almost quantitative correlation between nuclear dry mass and amount of ³H-thymidine incorporated into DNA. In order to determine the relative amount of ³H-thymidine per single cell grain counts were done by incident light photometry. They were corrected for the coincidence of -corpuscles as well as for the influence of -self absorption. A non-linear relation between nuclear dry mass and incorporation of ³H-thymidine per nucleus seems to be most probable.

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Studie im Rahmen der Assoziation Hämatologie EURATOM-GSF.

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Wir danken Herrn Prof. Dr. W. Maurer, Inst. für Medizinische Isotopenforschung der Universität Würzburg, für die Überlassung der Unterlagen zur Berechnung von - Absorption und - Selbstabsorption.     

Identification of tyrosine-phosphorylated colony-stimulating factor 1 (CSF-1) receptor and a 56-kilodalton protein phosphorylated in intact human cells in response to CSF-1

Colony-stimulating factor 1 (CSF-1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF-1, (the c-fms protein) is a protein-tyrosine kinase activated by the binding of CFS-1, the role of phosphorylation of cellular proteins in CSF-1 signal transduction is poorly understood. Therefore, we examined the CSF-1-stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c-fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF-1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170-kd protein, followed closely by the phosphorylation of a 56-kd protein, was observed in response to CSF-1. The 170-kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti-fms serum, consistent with its identity as the CSF-1 receptor. Although purified human macrophages that proliferate in culture in response to CSF-1 are not generally accessible, CSF-1 did stimulate the phosphorylation of a 56-kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF-1-stimulated cellular protein phosphorylation.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Auer-rod positive myelocytic leukemia cells in diffusion chambers: differentiation along the eosinophilic pathway

Auer-rod positive acute myelocytic leukemia (AML) blasts from a 33-year-old patient were cultured in diffusion chambers (DC) in order to test their differentiation potential. The cells were labeled with anti-human granulocyte anti-serum known to be negative for eosinophils, and evaluated using the unlabeled peroxidase-antiperoxidase (PAP) method. Parallel to a decline in the number of leukemic blasts there was an increase of up to 86% in the number of granulocytic cells belonging to the eosinophilic series. Auer-rod bodies were found in the eosinophilic cells even after 20 days in culture. Staining with anti-granulocyte antiserum failed to demonstrate positive cells at any time during DC culture. Based on the negative reaction with the anti-granulocytic antibodies already at an early stage of development evidence is provided for the existence of a progenitor cell exclusively committed to the eosinophilic pathway.     

Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture

Summary When Clostridium acetobutylicum was grown in continuous culture under glucose limitation at neutral pH and varying dilution rates the only fermentation products formed were acetate, butyrate, carbon dioxide and molecular hydrogen. The Y _glucose ^max and (Y _ATP ^max ) _gluc ^exp values were 48.3 and 23.8 dry weight/mol, respectively. Acetone and butanol were produced when the pH was decreased below 5.0 (optimum at pH 4.3). The addition of butyric acid (20 to 80 mM) to the medium with a pH of 4.3 resulted in a shift of the fermentation from acid, to solvent formation.A preliminary report of part of this work was presented at a symposium Trends in the Biology of Fermentations for Fuels and Chemicals held December 7–11, 1980, at Brookhaven National Laboratory, Upton, New York; Gottschalk and Bahl 1981     

Über die rationelle Milchpasteurisierung

Ohne ZusammenfassungDie Arbeit wurde dem R. Istituto Lombardo Scienze e Lettere am 5. Juni 1934 vorgelegt (Rend. 67, 671, 1934).     

Improvement of pneumonia and arthritis in Felty's syndrome by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF)

A 63-year old man with Felty's syndrome and pneumonia of unknown origin was treated with GM-CSF. Granulocyte counts increased and arthritis-related symptoms improved under GM-CSF. Pneumonia was treated effectively with antibiotics only during or after GM-CSF application. This suggests, that antibiotic-resistant infections can be treated effectively in patients with Felty's syndrome when granulocyte counts are raised by GM-CSF.