Gliding motility of Mycoplasma sp. nov. strain 163K.

The gliding movements of Mycoplasma sp. nov. strain 163K cells were characterized by photomicrographic and microcinematographic studies. The capability of gliding proved to be a very stable property of strain 163K. Cells were continuously moving, without interruption by resting periods, on glass as well as on plastic surfaces covered with liquid medium. Gliding cells always moved in the direction of their headlike structure; their course did not indicate any preference for a certain direction. Under appropriate growth conditions, cells showed linear and circular movements. Under inadequate conditions, cells glided in narrow circles or entered into zigzag trembling and tumbling movements. Organisms glided as single cells, in pairs, and in multicellular configurations. Movement patterns and gliding velocity were significantly affected by the cultivation and preparation time, the medium viscosity, and the storage and observation temperature. The number of passages on artificial media and the composition of the media used did not have a striking influence on gliding motility, but movements were effectively inhibited by homologous antiserum. The data obtained suggest that at least some of the structures associated with gliding are heat sensitive and located on the cell surface, that the gliding mechanism requires an intact energy metabolism, and, finally, that gliding motility is an extremely stable genetic property of Mycoplasma sp. nov. strain 163K.     

Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.     

Extracellular Signal-Regulated Kinase 7 (ERK7), a Novel ERK with a C-Terminal Domain That Regulates Its Activity, Its Cellular Localization, and Cell Growth

Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function.     

Functional context, biosynthesis, and genetic encoding of pyrrolysine

In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In Escherichia coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two molecules of lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.     

The surface layer of Mycoplasma mobile 163K and its possible relevance to cell cohesion and group motility

Mycoplasma mobile strain 163K tends to move in multicellular configurations, either as pairs or small groups of three or more cells, or as chain-like aggregations or microcolonies. Such wandering groups arise by transient association of independently moving cells. This behaviour of M. mobile was microscopically investigated and documented by sequences of microcinematographic pictures, as well as by photomicrographically recorded motility tracks. The presence of an extracellular slime layer was demonstrated in thin sections, by negative staining and by scanning electron microscopy. The possible association of this layer with the cohesive properties of the mycoplasma cells, enabling the formation of wandering groups, is discussed and a calculation of the magnitude of the cohesive force is provided.     

Effects of chronic exposure to insulin on lipid synthesis in 3T3-L1 fatty fibroblasts

Summary The acute effect of insulin on ³H incorporation into lipid from glucose was measured in 3T3-L1 fatty fibroblasts cultured with and without insulin at 10 µg/ml for 7 days. Basal lipid synthesis did not differ between control cells and cells treated chronically with insulin. There was no insulin stimulation in treated cells while ³H incorporation into lipid in control cells increased from a basal level of 1.39 to 3.85 nmol/dish/90 min with a maximally-stimulating concentration of insulin. This is the first study of 3T3-L1 fatty fibroblasts which describes a lack of acute insulin responsiveness in cells exposed chronically to insulin as compared to control cells.Abbreviations KRP buffer Kreb's Ringer phosphate buffer - BSA bovine serum albumin Dr. Pohl is the recipient of Research Career Development Award AM 00183.