Synthesis and structure of two polymeric barium-oxydiacetates: [Ba(oda) · H2O]n and [Ba(Hoda)2]n (H2oda: oxydiacetic acid)

The reaction of barium carbonate or hydroxide with oxydiacetic acid leads to the self-assembly of two barium oxydiacetate polymers in good yield: [Ba(oda) · H₂O]_n (1) and [Ba(Hoda)₂]_n (2). The products have been characterized by elemental analysis, IR, TGA and single crystal X-ray diffraction studies. The central barium atom in each mononuclear fragment is nine-coordinate in 1 and 10-coordinate in 2. These fragments are bridged by carboxylato groups in anti-anti conformation and through H-bonds bonding interactions forming complex 3D networks.     

Toxicity of cadmium in tobacco smoke: protection by antioxidants and chelating resins

The effects of cadmium, an environmental toxin present in tobacco smoke, were studied in vitro in human monocytes and compared to those of tobacco smoke. Overexpression of the 72 λkDa heat shock/stress protein Hsp70 and cell death occurred with a similar time-course and to a similar extent in human monocytes exposed to either cadmium or tobacco smoke. Cadmium and tobacco smoke-mediated toxicity were associated with a decrease in the cellular content of glutathione and ATP and the glutathione precursor N -acetyl- l -cysteine prevented both cadmium and tobacco smoke-mediated toxicity. Furthermore, tobacco smoke-mediated toxicity was prevented by pretreatment with the cadmium chelator resin Chelex-100, supporting the conclusion that cadmium plays a major role in tobacco smoke-mediated toxicity.     

Acidification of Soil in a Dry Land Winter Wheat-sorghum/corn-fallow Rotation in the Semiarid U.S. Great Plains

Soil pH is decreasing in many soils in the semiarid Great Plains of the United States under dry land no-till (NT) cropping systems. This study was conducted to determine the rate of acidification and the causes of the acidification of a soil cropped to a winter wheat (Triticum aestivum L.)-grain sorghum [Sorghum bicolor (L.) Moench]/corn (Zea mays L.)-fallow rotation (W-S/C-F) under NT. The study was conducted from 1989 to 2003 on soil with a long-term history of either continuous NT management [NT(LT)] (1962–2003) or conventional tillage (CT) (1962–1988) then converted to NT [NT(C)] (1989–2003). Nitrogen was applied as ammonium nitrate (AN) at a rate of 23 kg N ha⁻¹ in 1989 and as urea ammonium nitrate (UAN) at an average annual rate of 50 kg N ha⁻¹ from 1990 to 2003 for both NT treatments. Soil samples were collected at depth increments of 0–5, 5–10, 10–15, and 15–30 cm in the spring of 1989 and 2003. Acidification rates for the NT(LT) and NT(C) treatments were 1.13 and 1.48 kmol H⁺ ha⁻¹ yr⁻¹ in the 0–30 cm depth, respectively. The amount of CaCO₃ needed to neutralize the acidification is 57 and 74 kg ha⁻¹ yr⁻¹ for the NT(LT) and NT(C) treatments, respectively. A proton budget estimated by the Helyar and Porter [1989, Soil Acidity and Plant Growth, Academic Press] method indicated that NO₃⁻ leaching from the 30 cm depth was a primary cause of long-term acidification in this soil. Nitrate leaching accounted for 59 and 66% of the H⁺ from the acid causing factors for NT(LT) and NT(C) treatments, respectively. The addition of crop residues to the soil neutralized 62 and 47% of the acidity produced from the leaching of NO₃⁻, and 37 and 31% of the acid resulting from NO₃⁻ leaching and the other acid-causing constituents for the NT(LT) and NT(C) treatments, respectively. These results document that surface soils in dry land W-S/C-F rotations under NT are acidifying under current management practices. Improved management to increase nitrogen uptake efficiency from applied fertilizer would help reduce the rate of acidification. The addition of lime materials to prevent negative impacts on grain yields may be necessary in the future under current management practices. A contribution of the university of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal series No. 15120     

Regulation of class II gene expression in a murine pre-B cell line

Class II major histocompatibility complex (MHC) gene expression has been studied in an Abelson virus-transformed pre-B cell line R8, and its Ia-negative variant R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of trans-acting factors that can regulate class II gene expression. Furthermore, the T cell-derived lymphokine B cell stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.     

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.     

Extent of high-affinity iron transport systems in field isolates of rhizobia

A Uruguayan rhizobia collection (67 isolates) obtained from nodules of Medicago sativa, Melilotus albus, Medicago polymorpha, Trifolium subterraneum, Trifolium repens, Trifolium vesiculosum, Lotus corniculatus, Lotus subbiflorus, Lotus pedunculatus, Ornithopus sp. and Adesmia sp. has been examined to assess the occurrence of high affinity iron uptake systems. CAS (Chrome-azurol S)-assay results suggested that most of the free-living form of these microsymbionts may produce siderophores. The highest siderophore production was observed among Medicago and Trifolium microsymbionts whereas no siderophore expression or moderate positive results were found among Lotus microsymbionts; suggesting that microsymbionts of legumes growing on neutral or alkaline soils may express in vitro enhanced siderophore production. Electrophoretic patterns of outer-membrane protein enriched fractions revealed that iron-limited microsymbionts of Medicago sativa, Lotus corniculatus, Lotus subbiflorus, Trifolium repens, Trifolium subterraneum and Ornithopus sp. produced high molecular weight proteins (ranging from 64 to 94 kDa) compared to cells grown in iron-sufficient media.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.     

Midostaurin upregulates eNOS gene expression and preserves eNOS function in the microcirculation of the mouse

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is a powerful vasodilator and possesses vasoprotective effects. Therefore, augmentation of eNOS expression and -activity by pharmacological means could provide protection against cardiovascular disease. However, this concept has been questioned recently, because in several disease models, eNOS upregulation was associated with a dysfunctional enzyme (referred to as eNOS uncoupling). In contrast, the present study demonstrates that an eNOS gene expression-enhancing compound with additional protein kinase C (PKC) inhibitory properties can upregulate eNOS while preserving its enzymatic function. Apolipoprotein E-knockout mice were treated for 7 days with midostaurin (4'-N-benzoyl staurosporine, compound CGP 41251, 50-125 mg/kg/day), a PKC inhibitor previously shown to increase eNOS expression and NO production in cultured human endothelial cells. Midostaurin treatment enhanced eNOS mRNA expression (RNase protection assay) in mouse aorta, kidney, and heart in a dose-dependent fashion. In the dorsal skinfold microcirculation, midostaurin produced an arteriolar vasorelaxation (intravital microscopy), which could be prevented by the NOS inhibitor L-NAME, indicating that the upregulated eNOS remained functional. In organ chamber experiments, the aorta from midostaurin-treated mice showed an enhanced NO-mediated relaxation in response to acetylcholine. Accordingly, serum levels of nitrite/nitrate (NO-Analyzer) were increased, and the production of reactive oxygen species in the aorta (L-012 chemiluminescence) was reduced by midostaurin. Thus, in mice in vivo, midostaurin treatment results in enhanced expression of eNOS with preserved enzyme function and enhanced production of bioactive NO. Given the beneficial effects of endothelial-derived NO, vasoprotective and anti-atherosclerotic effects are likely to ensue.