Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.     

Rat model of the human "Triton" tumor: direct genetic evidence for the myogenic differentiation capacity of schwannoma cells using the mutant neu gene as a cell lineage marker

Spontaneous myogenic differentiation was observed in 2 out of 15 cases when cells from schwannomas induced in the offspring of BDIX rats by transplacental exposure to N-ethyl-N-nitrosourea (EtNU) were grown in monolayer culture following fluorescence-activated cell sorting with monoclonal antibody (Mab) 217c. Myotubes and numerous mononucleated cells no longer expressed the Schwann cell antigens 217c and S-100 protein, but rather revealed the presence of desmin, the alpha-sarcomeric form (alpha-sr) of actin, and the cell surface antigen specified by Mab RB21-7, a 250 kD glycoprotein sharing an epitope with the neural cell adhesion molecule (N-CAM). Subcutaneous reimplantation of such cells into syngeneic animals led to the appearance of tumors composed of both S-100 positive Schwann cells and desmin and alpha-sr-actin positive rhabdomyoblasts, thus closely resembling the human "Triton" tumor. With the use of the polymerase chain reaction and allele-specific oligonucleotide hybridization, DNA isolated from individual myotubes was analyzed for the presence of a T----A transversion mutation at nucleotide 2012 of the neu gene, which is diagnostic of EtNU-induced rat schwannomas. All of the amplified DNA isolates contained the mutant neu allele, thus providing direct genetic proof for the capacity of mammalian neuroectodermal cells for myogenic differentiation.     

Thermogenic effect of adrenaline: interaction with insulin

The contribution of insulin (3.6 pmol.kg body mass-1.min-1) to adrenaline-induced (0.164 nmol.kg fat free mass-1.min-1) thermogenesis was studied in ten postabsorptive healthy volunteers using two sequential protocols. Variables considered were oxygen consumption as well as carbon dioxide production, heart rate, blood pressure, plasma concentrations of glucose, insulin, glycerol, free fatty acids, beta-HO-butyrate and lactate. Adrenaline increased plasma concentrations of glucose, glycerol, free fatty acids, and beta-HO-butyrate, and heart rate and metabolic rate during normo-insulinaemia [61.3 (SEM 6.6) pmol.l-1]. Similar effects were observed during hyperinsulinaemia [167.9 (SEM 18.7) pmol.l-1], but the effect of adrenaline on oxygen consumption was reduced. On average, metabolic rate increased by 12.9% during normo-insulinaemia and by 8.9% during hyperinsulinaemia. We concluded that relative hyperinsulinaemia resulted in decreased adrenaline-induced thermogenesis and therefore increased whole body anabolism.     

Oxidative phosphorylation in membrane vesicles of a gram-positive methylotroph

Membrane preparations, capable of high rates of respiration-linked ATP synthesis, have been obtained from a gram-positive methylotrophic bacterium Bacillus sp. MGA3. NADH, succinate, reduced TMPD and methanol were shown to be suitable substrates for the oxidative phosphorylation. Esterification of orthophosphate was dependent on electron transfer, as evidenced by the requirement for both substrate and oxygen. Phosphorylation was also dependent on ADP and was destroyed by boiling the membrane preparation. The phosphorylation was markedly uncoupled by carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone (CCCP) and was inhibited by N,N-dicyclohexylcarbodiimide (DCCD). KCN caused strong inhibition of substrate oxidation as well as phosphorylation for all substrates tested. Rotenone, amytal and antimycin A caused inhibition when NADH or methanol were used as substrates. Antimycin A inhibited respiration and ATP synthesis with succinate as substrate and had no effect on ascorbate —N,N,N,N-tetramethyl-p-phenylenediimide (TMPD) oxidation by membrane preparations of Bacillus sp. MGA3. P/O ratios determined were 2.4 with NADH, 1.7 with succinate and 0.8 with reduced TMPD. The measured P/O ratio with methanol-oxidizing system was similar to that with NADH (about 2.4).Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - TMPD N,N,N,N-tetramethyl-p-phenylenediimide - Q ubiquinone Q     

Cytogenetic and molecular characterization of a newly established neuroblastoma cell line LS

Summary A new human neuroblastoma cell line (LS) that originated from an abdominal tumor of a 16-month-old girl is presented; it was classified, according to Evans, as being stage III. Morphological (dense-core particles) and biochemical characteristics (dopamine--hydroxylase, acetylcholinesterase, neuron-specific-enolase) confirmed the diagnosis. In addition to a slightly variable modal chromosome number of 48 or 49 (because of marker-chromosomes and autosomal trisomies), cytogenetic analysis revealed two constantly appearing chromosomes with homogeneously stained regions (HSR's). The karyo-type remained constant over 50 passages in vitro [49,XX, –12,+der5, + 17,+mar1,+mar2]. Double minutes were a rare phenomenon and appeared only in a few metaphases. In situ hybridization showed that some of the HSR's consisted of amplified N-myc copies. The distribution of the N-myc copies according to in situ hybridization signals along the HSR's was compared with the data of Southern and Northern blotting analyses.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.     

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight⁻¹ at D = 0.1 h⁻¹ to 2 U mg of cell dry weight⁻¹ at D = 0.8 h⁻¹. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.     

Role of chemical concentration and second carbon sources in acclimation of microbial communities for biodegradation.

A study was conducted to determine the role of concentration of the test chemical, of a second organic compound, and of mutation in the acclimation period before the mineralization of organic compounds in sewage. The acclimation period for the mineralization in sewage of 2 micrograms of 4-nitrophenol (PNP) per liter increased from 6 to 12 days in the presence of 10 mg of 2,4-dinitrophenol per liter. The extension of the acclimation period was equivalent to the time required for mineralization of 2,4-dinitrophenol. In contrast, the time for acclimation for the degradation of 2 micrograms of PNP per liter was reduced when 10 or 100 mg of phenol per liter was added. Lower phenol levels increased the acclimation period to 8 days. The length of the acclimation period for PNP mineralization decreased as the initial concentration of PNP increased from 2 micrograms to 100 mg/liter. The acclimation period for phenol mineralization was lengthened as the phenol concentration increased from 100 to 1,400 mg/liter. The length of the acclimation period for PNP and phenol biodegradation was reproducible, but it varied among replicates for the biodegradation of other nitro-substituted compounds added to sewage or lake water, suggesting that a mutation was responsible for acclimation to these other compounds. The acclimation period may thus reflect the time required for the destruction of toxins, and it also may be affected by the concentration of the test compound or the presence of other substrates.