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The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewhere
Cytochrome oxidasewas prepared from Neurospora crassa by chromatography on oleyl polymethacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation oflabelled amino acids into the single polypeptideswas investigated after a pulse labelling in the absence and presence of chloramphenicol, and afterwashing out the inhibitor. Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90-95%• while labeHing of the whole membrane protein was inhibited only 30%• Mter washing out the inhibitor and further growth of the cells. the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a. small increase in radioactivity. It is concluded that the four small-sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol.
no abstract available
The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides.
The responsiveness to IL-4 with and without costimulation with anti-IgM antibodies or phorbolester was studied in 35 cases of low grade non-Hodgkin Iymphoma by analyzing enhancement of CD23 and HLA dass li expression. The predominant phenotype responds directly to IL-4. Separate differentiation states can be distinguished according to coordinate or differential upregulation of CD23 and HLA dass II molecules by IL-4 alone, and differences in responsiveness to anti-IgM antibodies. A particular subgroup of B-lymphoma cells defines a separate stage of B-eeil differentiation. They fail to express high affinity binding sites for IL-4 and accordingly do not respond to IL-4- mediated signals. Cross-linking membrane lgM receptors or direct activation of protein kinase C via phorbolester induces IL-4 receptor expression and subsequent IL-4 reactivity.
Human interleukin-4 possesses two distinct sites for receptor activation. A signaHing site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of böth Arg121 and Tyr124 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y124D]interleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cellline with a K\(_i\) value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleuk.in-13- induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Rm the extracellular domain of the interleuk.in-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system.