Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Rapid Genotyping of Mice with Hemoglobinopathies and Globin Transgenes

The hematology of the laboratory mouse has beenwell characterized. Normal genetic differences at thealpha- and beta-globin gene loci serve as useful markersfor a wide variety of types of experimental studies. There are a number of naturallyoccurring or induced mutations that disrupt globinexpression and produce thalassemic phenotypes. Inaddition, much has been learned of the workings of theglobin locus control region from studies of transgenicmice, including those with mutations induced by targetedsite-specific modifications. After a new mutation ortransgene has been created, it must be maintained in living mice, and the genotypes of theoffspring must be ascertained. While it is possible todetermine genotypes by DNA analyses, such assays aretime consuming and relatively expensive. An osmoticchallenge test -- originally developed for thegenotyping of large-deletion alpha-thalassemia mutationsin mice -- has proven useful in detecting bothsevere and milder alpha- and beta-thalassemias, as wellas some transgenic genotypes in mice carrying human globin genes.Reliable genotyping can, in some cases, be completedwithin a few minutes with minimal expense.Quantification of red cell fragility for a variety ofthalassemic and transgenic mice is described here, alongwith a simplified test suitable for rapid, routinegenotyping. The osmotic challenge test is perfectlyreliable for distinguishing genotypes that causesignificantly decreased release of hemoglobin from the redcells, but it is also useful for some of the conditionsin which overall erythrocyte osmotic fragility isessentially normal.     

The Saccharomyces cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in vivo

Mismatch repair proteins act during double-strand break repair (DSBR) to correct mismatches in heteroduplex DNA, to suppress recombination between divergent sequences, and to promote removal of nonhomologous DNA at DSB ends. We investigated yeast Msh2p association with recombination intermediates in vivo using chromatin immunoprecipitation. During DSBR involving nonhomologous ends, Msh2p localized strongly to recipient and donor sequences. Localization required Msh3p and was greatly reduced in rad50delta strains. Minimal localization of Msh2p was observed during fully homologous repair, but this was increased in rad52delta strains. These findings argue that Msh2p-Msh3p associates with intermediates early in DSBR to participate in the rejection of homeologous pairing and to stabilize nonhomologous tails for cleavage by Rad1p-Rad10p endonuclease.     

Increased Renal Iron Accumulation in Hypertensive Nephropathy of Salt-Loaded Hypertensive Rats

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.     

Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.     

FINE STRUCTURE OF THE OCTOPUS RETINA

The fine structure of the visual and the supporting cells and of the blood capillaries in the octopus retina is described. Lamellated structures contained in the proximal segment of the visual cell consist of compact arrays of dense membranes each of which is quintuple-layered and divides at its margins into two thinner sheets or membranes which are connected directly with the agranular or granular endoplasmic reticulum. Proximal to the deeper extremities of the rhabdomeres, the lateral plasma membranes of two adjoining visual cells contact each other forming a quintuple-layered compound membrane, which results in occlusion of the intercellular space. The central layer of the compound membrane is of high density, so that the membrane, as a whole, appears to be a single thick layer at low magnifications. The supporting cells are connected with the neighboring visual cells by two types of junctions. Long slender processes extend from the supporting cells to the surface of the retina through narrow spaces among the distal segments of the visual cells. The capillary endothelial cells are characterized by luminal surfaces irregularly contoured and by lateral surfaces elaborately interdigitated. The functional significance of the close contact between adjoining visual cells is discussed.     

Interleukin-18 (IL-18) and infectious diseases, with special emphasis on diseases induced by intracellular pathogens

Interleukin-18 (IL-18) is a novel cytokine mainly produced by activated macrophages. IL-18 was originally called interferon-gamma inducing factor, due to its action in inducing IFN-gamma secretion from Th1 cells, NK cells and NKT cells. It has been reported that IL-18 may play important roles in various diseases including cancer and infectious diseases. This review deals with the roles of IL-18 in infectious diseases, with special emphasis on IL-18 in infectious diseases caused by intracellular pathogens including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes and Salmonella typhimurium.