Fungal Catabolism of Crown Gall Opines

This study was conducted to determine the capacities of 37 fungi to utilize various crown gall opines as their sole carbon and nitrogen source. One strain of Fusarium solani, two of Cylindrocarpon destructans, and six of Cylindrocarpon heteronema catabolized octopine, mannopine, octopinic acid, succinamopine, or a combination of these opines. One C. heteronema and one Fusarium dimerum strain grew only on succinamopine. None of the fungal isolates had the ability to grow on nopaline. The catabolism of opines by fungi was confirmed by the disappearance of the opine from the growth medium and by an increase in final mycelial dry weight with rising initial concentration of test substrate. This study thus shows that the catabolism of opines is not restricted to bacteria.     

Effects of ethylene on the orientation of microtubules and cellulose microfibrils of pea epicotyl cells with polylamellate cell walls

Summary Following a 5 hours ethylene treatment, cortical cells of Pea (Pisum sativum L. var Alaska) epicotyl third internode showed a change in the orientation of both microtubules near the plasma membrane and recently deposited cellulose microfibrils. Control cortical cells had mostly transverse microtubules. The ratio of the average frequency of transverse to longitudinal microtubules was 6.0. After 5 hours of ethylene treatment, cortical cells had mostly longitudinal microtubules, with the ratio of transverse to longitudinal microtubules equal to 0.1. Epidermal cells were more variable than cortical cells with regard to the frequency of longitudinal and transverse microtubules. Observation of cortical cell walls in conventionally stained thin sections revealed that recent deposition of microfibrils had been primarily transverse in almost all of the control cortical cells sampled. In contrast, more than half of the ethylene-treated cortical cells had recent deposition oriented primarily longitudinally. This change in microtubule and microfibril orientation may be early enough to constitute the primary effect of ethylene leading to radial cell expansion.Research supported by NSF grant PCM 78-03244, A1, 2 to PBG and by a Research Corporation grant to WRE.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

In vitro uptake of particles by lysosomes

Isolated rat liver lysosomes were incubated with Percoll particles in vitro at 25 and 37 °C. On morphological examination the incubated lysosomes contain vesicles some of which enclose Percoll particles, indicating that invagination of the lysosomal membrane occurs in vitro by means of microautophagy. Vesiculation occurs by formation of flaplike processes or cuplike invaginations. At later time points of incubation Percoll particles can be seen free in the lysosomal matrix indicating rupture or digestion of the vesicular membrane. The uptake of isotopically pre-labelled Percoll particles increases with incubation time and temperature.It is concluded that lysosomes show microautophagic activity in vitro and that this may be a mechanism for degradation of soluble cytoplasmic proteins also in vivo.     

Lysosomal uptake of isolated cell organelles microinjected into HeLa cells

Lysosomes and microsomes were isolated from rat liver and microinjected into the cytoplasm of HeLa cells. The fate of the transplanted organelles and their effects on the recipient cells were followed in the electron microscope at various time intervals after administration. Needle injection with buffer or sucrose did not seem to evoke any ultrastructural alterations, such as induced autophagy or other signs of sublethal cell injury. Recipients of microinjected cell organelles elicited a rapid and conspicuous increase in membrane-bounded cytoplasmic vacuoles, concomitant with the disappearance of the injected material. Golgi complexes became abundant with many small vesicles clustering around their cisternae. The volume density of the lysosomal compartment increased 2-3-fold after organelle injection as compared with control-injected (0.3 M sucrose) or noninjected cells. Our preliminary results show that isolated cell organelles can be microinjected into cells n culture and indicate that the microinjected organelles were segregated from the cytoplasm into membrane-bounded vacuoles probably through autophagolysosome formation. Thus, this technique offers an additional approach for studies on the segregation and degradation of cell organelles in somatic cells and may enable more detailed analyses on the mechanisms of autophagic sequestration of specific cell organelles.     

MKR mice are resistant to the metabolic actions of both insulin and adiponectin: discordance between insulin resistance and adiponectin responsiveness

Most rodent models of insulin resistance are accompanied by decreased circulating adiponectin levels. Adiponectin treatment improves the metabolic phenotype by increasing fatty acid oxidation in skeletal muscle and suppressing hepatic glucose production. Muscle IGF-I receptor (IGF-IR)-lysine-arginine (MKR) mice expressing dominant-negative mutant IGF-IRs in skeletal muscle are diabetic with insulin resistance in muscle, liver, and adipose tissue. Adiponectin levels are elevated in MKR mice, suggesting an unusual discordance between insulin resistance and adiponectin responsiveness. Therefore, we investigated the metabolic actions of adiponectin in MKR mice. MKR and ob/ob mice were treated both acutely (28 microg/g) and chronically (for 2 wk) with full-length adiponectin. Acute hypoglycemic effects of adiponectin were evident only in ob/ob mice but not in MKR mice. Chronic adiponectin treatment significantly improved both insulin sensitivity and glucose tolerance in ob/ob but not in MKR mice. Adiponectin receptor mRNA levels and adiponectin-stimulated phosphorylation of AMPK in skeletal muscle and liver were similar among MKR, wild-type, and ob/ob mice. Thus MKR mice are adiponectin resistant despite normal expression of adiponectin receptors and normal AMPK phosphorylation in muscle and liver. MKR mice may be a useful model for dissecting relationships between insulin resistance and adiponectin action in regulation of glucose homeostasis.     

25-Hydroxyvitamin D depletion does not exacerbate MPTP-induced dopamine neuron damage in mice

Recent clinical evidence supports a link between 25-hydroxyvitamin D insufficiency (serum 25-hydroxyvitamin D [25(OH)D] levels <30 ng/mL) and Parkinson's disease. To investigate the effect of 25(OH)D depletion on neuronal susceptibility to toxic insult, we induced a state of 25(OH)D deficiency in mice and then challenged them with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We found there was no significant difference between control and 25(OH)D-deficient animals in striatal dopamine levels or dopamine transporter and tyrosine hydroxylase expression after lesioning with MPTP. Additionally, we found no difference in tyrosine hydroxylase expression in the substantia nigra pars compacta. Our data suggest that reducing 25(OH)D serum levels in mice has no effect on the vulnerability of nigral dopaminergic neurons in vivo in this model system of parkinsonism.     

Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway

In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.