Bacterial mitotic machineries

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the ParM protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.     

History and the global ecology of squamate reptiles

The structure of communities may be largely a result of evolutionary changes that occurred many millions of years ago. We explore the historical ecology of squamates (lizards and snakes), identify historically derived differences among clades, and examine how this history has affected present-day squamate assemblages globally. A dietary shift occurred in the evolutionary history of squamates. Iguanian diets contain large proportions of ants, other hymenopterans, and beetles, whereas these are minor prey in scleroglossan lizards. A preponderance of termites, grasshoppers, spiders, and insect larvae in their diets suggests that scleroglossan lizards harvest higher energy prey or avoid prey containing noxious chemicals. The success of this dietary shift is suggested by dominance of scleroglossans in lizard assemblages throughout the world. One scleroglossan clade, Autarchoglossa, combined an advanced vomeronasal chemosensory system with jaw prehension and increased activity levels. We suggest these traits provided them a competitive advantage during the day in terrestrial habitats. Iguanians and gekkotans shifted to elevated microhabitats historically, and gekkotans shifted activity to nighttime. These historically derived niche differences are apparent in extant lizard assemblages and account for some observed structure. These patterns occur in a variety of habitats at both regional and local levels throughout the world.     

Induction of SM-alpha-actin expression by mechanical strain in adult vascular smooth muscle cells is mediated through activation of JNK and p38 MAP kinase

Mechanical forces have direct effects on the growth and differentiation of vascular smooth muscle. The goal of this study was to examine the effects of cyclic mechanical strain on expression of smooth muscle-alpha-actin (SM-alpha-actin), a marker for the differentiated state of vascular smooth muscle, in cultured rat aortic smooth muscle cells (VSMC). Cells grown on dishes coated with either laminin or pronectin were subjected to mechanical strain and effects on expression of SM-alpha-actin were evaluated using the Flexercell Strain Unit. Application of mechanical strain to cells in full media increased SM-alpha-actin protein expression and promoter activity. This was not associated with any effect on growth. Mechanical strain increased activity of all three members of the MAP kinase family (ERKs, JNKs, and p38 MAP kinase), with similar kinetics. Inhibition of either JNKs or p38 MAP kinase blocked the strain-induced increase in SM-alpha-actin promoter activity, and expression of constitutively active forms of JNK or MKK6, a p38 kinase, increased promoter activity. These studies indicate that in adult VSMC, mechanical strain leads to increased expression of smooth muscle markers, resulting in a more contractile phenotype.     

Pulse radiolysis,electron paramagnetic resonance spectroscopy and theoretical calculations of caffeic acid oligomer radicals

Seven representative compounds isolated from Salvia officinalis, among them caffeic acid, the dimer rosmarinic acid and oligomers of caffeic acid, were investigated with regard to their antioxidant potential both expressed by the radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic investigation revealed very high rate constants with both hydroxyl and azide radicals. Evidence from kinetic modelling calculations suggested unusual complex behavior due to the presence of both O(4)- and O(3)-semiquinones and - in two cases - formation and decay of a hydroxyl radical adduct at the vinyl side chain. EPR spectroscopy studies, which included dihydrocaffeic acid as a model for the saturated side chains of the oligomers, confirmed that the radical structures after oxidation in slightly alkaline solutions are representing dissociated O(4)-semiquinones. While according to calculations based on hybrid density-functional theory the other radical structures are valid intermediates, they cannot be observed except by pulse radiolysis due to their fast decay.     

Male axillary extracts contain pheromones that affect pulsatile secretion of luteinizing hormone and mood in women recipients

Human underarm secretions, when applied to women recipients, alter the length and timing of the menstrual cycle. These effects are thought to arise from exposure to primer pheromones that are produced in the underarm. Pheromones can affect endocrine (primer) or behavioral (releaser) responses, provide information (signaler), or perhaps even modify emotion or mood (modulator). In this study, we extracted underarm secretions from pads worn by men and placed the extract under the nose of women volunteers while monitoring serum LH and emotion/mood. Pulses of LH are excellent indicators of the release of GnRH from the brain's hypothalamus. In women, the positive influence of GnRH on LH affects the length and timing of the menstrual cycle, which, in turn, affects fertility. Here we show that extracts of male axillary secretions have a direct effect upon LH-pulsing and mood of women. In our subjects, the putative male pheromone(s) advanced the onset of the next peak of LH after its application, reduced tension, and increased relaxation. These results demonstrate that male axillary secretions contain one or more constituents that act as primer and modulator pheromones.     

The glutathione synthetase of Schizosaccharomyces pombe is synthesized as a homodimer but retains full activity when present as a heterotetramer

Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family.     

A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis

Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.     

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping "ladders" of Bax and Bak complexes in multiples of approximately 21 and approximately 23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.     

Keratin 8 protection of placental barrier function

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8-/- embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8-/- lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8-/- embryos. Finally, we show that K8-/- concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8-/- embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.     

Nanometer targeting of microtubules to focal adhesions

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP-CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.