Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

An unusual cation channel mediates photic control of locomotion in Drosophila

A unique family of putative ion channels that are related to voltage-gated sodium and calcium channels has been identified in genomic and cDNA studies of metazoans. Aside from evidence for expression of family members in the nervous system, little is known about the operation of the channel or its functional significance. In the present study, this conserved family's sole Drosophila member, a gene known both as CG1517 and as Dmalpha1U, is shown to correspond to the narrow abdomen (na) gene and is the locus of a set of mutations that affect sensitivity to anesthetics. Immunohistochemistry of adult heads reveals that the channel is expressed in the neuropil of the central complex and optic lobe; expression is severely depressed in the mutants. In addition to previously described defects, the mutant phenotype is demonstrated here to include dysfunction in the coupling between light and locomotor behavior. Most dramatically, mutant flies have an inversion of relative locomotor activity in light versus dark. The involvement of the channel in daily rhythms of the fruit fly is especially provocative because the human ortholog lies in a candidate region linked to bipolar disorder, a disease frequently associated with altered diurnal behavior.     

Comparative analysis of quantitative trait loci controlling glucosinolates,myrosinase and insect resistance in Arabidopsis thaliana

Evolutionary interactions among insect herbivores and plant chemical defenses have generated systems where plant compounds have opposing fitness consequences for host plants, depending on attack by various insect herbivores. This interplay complicates understanding of fitness costs and benefits of plant chemical defenses. We are studying the role of the glucosinolate-myrosinase chemical defense system in protecting Arabidopsis thaliana from specialist and generalist insect herbivory. We used two Arabidopsis recombinant inbred populations in which we had previously mapped QTL controlling variation in the glucosinolate-myrosinase system. In this study we mapped QTL controlling resistance to specialist (Plutella xylostella) and generalist (Trichoplusia ni) herbivores. We identified a number of QTL that are specific to one herbivore or the other, as well as a single QTL that controls resistance to both insects. Comparison of QTL for herbivory, glucosinolates, and myrosinase showed that T. ni herbivory is strongly deterred by higher glucosinolate levels, faster breakdown rates, and specific chemical structures. In contrast, P. xylostella herbivory is uncorrelated with variation in the glucosinolate-myrosinase system. This agrees with evolutionary theory stating that specialist insects may overcome host plant chemical defenses, whereas generalists will be sensitive to these same defenses.     

Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.     

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.     

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated.     

Manipulation of dendritic cells as an approach to improved outcomes in transplantation

At the time of organ transplantation, a variety of non-parenchymal cells are transplanted simultaneously with the allograft. Recognition of the importance of these cells as potential immunostimulatory cells lead to the concept of 'passenger leukocytes' as the principal instigators of rejection. Passenger leukocytes include interstitial dendritic cells (DCs) and blood-derived monocytes/macrophages. As investigators have discovered the significance of DCs in influencing graft outcome, so have they begun to determine the best ways to influence DCs themselves. This review discusses the role of DCs in transplantation and then focuses on three different approaches for manipulating DCs to improve allograft survival: (1) targeting of chemokines involved in DC migration, (2) pharmacological arrest of DC maturation, and (3) genetic engineering of DCs.     

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells

Antigen-mediated activation of mast cells results in Ca²⁺-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca²⁺ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response .     

Investigating plant-plant interference by metabolic fingerprinting

New analytical developments in post-genomic technologies are being introduced to the field of plant ecology. FT-IR fingerprinting coupled with chemometrics via cluster analysis is proposed as a tool for correlating global metabolic changes with abiotic or biotic perturbation and/or interactions. The current study concentrates on detecting chemical responses by inter-species competition between a monocotyledon Brachypodium distachyion and a dicotyledon Arabidopsis thaliana. Growth analysis of 42 days old plants showed differences in both species under competition. Clear changes in the FT-IR metabolic fingerprints of B. distachyion in competition with A. thaliana were observed, whilst there were no apparent chemical differences in the A. thaliana plant tissues. This study demonstrates the power of this approach in detecting changes in the global metabolic profiles of plants in response to biotic interactions, and we believe FT-IR is appropriate for rapid screening (10 s per sample) prior to targeted metabolite analyses.