Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.     

Nitrogen processing in the hyporheic zone of a pastoral stream

The distribution of nitrogen-transforming processes, and factors controlling their rates, were determined within the hyporheic zone of a lowland stream draining agricultural land. In the field, physicochemical parameters were measured along a 10m-long hyporheic flow line between downwelling and upwelling zones. Sediment cores were retrieved from the stream bed surface, and from 20, 40 and 60cm deep in each zone, and in the laboratory, water from the corresponding depth was percolated through each core at the natural flow rate. Concentrations of nitrogen species and oxygen were measured before and after flow through each core. Denitrification was measured using a ¹⁵N-nitrate tracer. Shallow and downwelling zone samples were clearly distinct from deeper and upwelling zone samples in terms of physicochemical conditions, microbial processes and factors controlling nitrogen processing. Denitrification was highest in surface and downwelling zone cores, despite high oxygen levels, probably due to high pore-water nitrate concentrations in these cores and isolation of the denitrifying bacteria from oxygen in the bulk water by the hyporheic biofilms. Denitrification was limited by oxygen inhibition in the downwelling group, and by nitrate availability in the upwelling group. Strong evidence indicated that dissimilatory nitrate reduction to ammonium, occurred in almost all cores, and outcompeted denitrification for nitrate. In contrast, nitrification was undetectable in all but two cores, probably because of intense competition for oxygen. Field patterns and lab experiments indicated that the hyporheic zone at this moderately N-rich site is a strong sink for nitrate, fitting current theories that predict where hyporheic zones are nitrate sinks or nitrate sources.     

Quinazolinone fungal efflux pump inhibitors. Part 2: In vitro structure-activity relationships of (N-methyl-piperazinyl)-containing derivatives

Structure-activity relationships of a novel series of fungal efflux pump inhibitors with respect to potentiation of the activity of fluconazole against strains of Candida albicans and Candida glabrata over-expressing ABC-type efflux pumps are systematically explored.     

Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.     

Ethanol, fruit ripening, and the historical origins of human alcoholism in primate frugivory

Ethanol is a naturally occurring substance resulting from thefermentation by yeast of fruit sugars. The association betweenyeasts and angiosperms dates to the Cretaceous, and dietaryexposure of diverse frugivorous taxa to ethanol is similarlyancient. Ethanol plumes can potentially be used to localizeripe fruit, and consumption of low-concentration ethanol withinfruit may act as a feeding stimulant. Ripe and over-ripe fruitsof the Neotropical palm Astrocaryum standleyanum contained ethanolwithin the pulp at concentrations averaging 0.9% and 4.5%, respectively.Fruit ripening was associated with significant changes in color,puncture resistance, sugar, and ethanol content. Natural consumptionrates of ethanol via frugivory and associated blood levels arenot known for any animal taxon. However, behavioral responsesto ethanol may have been the target of natural selection forall frugivorous species, including many primates and the hominoidlineages ancestral to modern humans. Pre-existing sensory biasesassociating this ancient psychoactive compound with nutritionalreward might accordingly underlie contemporary patterns of alcoholconsumption and abuse.     

Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition

MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.     

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.     

The cytoplasmic domain of the low density lipoprotein (LDL) receptor-related protein,but not that of the LDL receptor,triggers phagocytosis

The macrophage LDL receptor and LDL receptor-related protein (LRP, CD91) mediate the phagocytic-like uptake of atherogenic lipoproteins and apoptotic cells, yet the structural basis of their phagocytic functions is not known. To address this issue, we transfected macrophages with chimeric proteins containing the cytoplasmic tails and transmembrane regions of the LDL receptor or LRP and the ectodomain of CD2, which can bind non-opsonized sheep red blood cells (SRBCs). Macrophages expressing receptors containing the LDL receptor domains were able to bind but not internalize SRBCs. In contrast, macrophages expressing receptors containing the cytoplasmic tail of LRP were able to bind and internalize SRBCs. Chimeras in which the LRP cytoplasmic tail was mutated in two di-leucine motifs and a tyrosine in an NPXYXXL motif were able to endocytose anti-CD2 antibody and bind SRBCs, but SRBC phagocytosis was decreased by 70%. Thus, the phagocytic-like functions of LRP, but not those of the LDL receptor, can be explained by the ability of the LRP cytoplasmic tail to trigger phagocytosis. These findings have important implications for atherogenesis and apoptotic cell clearance and for a fundamental cell biological understanding of how the LDL receptor and LRP function in internalization processes.     

A role for alphaV integrin subunit in TGF-beta-stimulated osteoclastogenesis

TGF-beta increases bone resorption in vivo and greatly increases osteoclast formation stimulated by receptor activator of NF-kappaB ligand (RANKL) in vitro. TGF-beta does not independently affect the differentiation state of RAW264.7 preosteoclasts, but increases cell attachment to vitronectin. This effect is mediated by increased expression of alphaV integrin subunit mRNA and protein. Concomitant with induction of osteoclast differentiation, RANKL causes relocation of alphaV to focal sites in the cell. This effect is potentiated by TGF-beta. Integrin blockade disrupts both attachment to vitronectin and RANKL-induced osteoclast formation, but culture on vitronectin has little effect. Ectopic expression of alphaV stimulates multinucleation of RAW264.7 cells and increases the number of osteoclasts formed in the presence of RANKL. These data suggest that TGF-beta potentiates RANKL-induced osteoclast formation, in part by increased expression of the alphaV integrin subunit, which may contribute to cell fusion.