Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution

Since the discovery of the first human neocentromere in 1993, these spontaneous, ectopic centromeres have been shown to be an astonishing example of epigenetic change within the genome. Recent research has focused on the role of neocentromeres in evolution and speciation, as well as in disease development and the understanding of the organization and epigenetic maintenance of the centromere. Here, we review recent progress in these areas of research and the significant insights gained.     

Intraosseous angiosarcoma with secondary aneurysmal bone cysts presenting as an elusive diagnostic challenge

Angiosarcoma of bone is an exceedingly rare primary bone malignancy that can present as an aggressive osteolytic lesion. Histological diagnosis can be extremely challenging, as the pathological features often resemble that of aneurysmal bone cysts. We report an interesting and peculiar case of an intraosseous angiosarcoma that presented as a diagnostic dilemma and discuss the relevant radiological and pathologic findings.     

The impact of HLA-DRB1 genes on extra-articular disease manifestations in rheumatoid arthritis

The objective of this study was to examine HLA-DRB1 and HLA-DQB1 genotypes in patients with severe extra-articular rheumatoid arthritis (ExRA) and to compare them with the genotypes of rheumatoid arthritis (RA) patients without extra-articular manifestations. Patients with severe ExRA were recruited from a large research database of patients with RA, from two cohorts of prevalent RA cases, and from a regional multicenter early RA cohort. Cases with ExRA manifestations (n = 159) were classified according to predefined criteria. Controls (n = 178) with RA but no ExRA were selected from the same sources. Cases and controls were matched for duration of RA and for clinical center. PCR based HLA-DRB1 and HLA-DQB1 genotyping was performed using the Biotest SSP kit, with additional sequencing in order to distinguish DRB1*04 subtypes. Associations between alleles and disease phenotypes were tested using multiple simulations of random distributions of alleles. There was no difference in global distribution of HLA-DRB1 and HLA-DQB1 alleles between patients with ExRA and controls. DRB1*0401 (P = 0.003) and 0401/0401 homozygosity (P = 0.002) were more frequent in Felty's syndrome than in controls. The presence of two HLA-DRB1*04 alleles encoding the shared epitope (SE) was associated with ExRA (overall odds ratio 1.79, 95% confidence interval 1.04-3.08) and with rheumatoid vasculitis (odds ratio 2.44, 95% confidence interval 1.22-4.89). In this large sample of patients with ExRA, Felty's syndrome was the only manifestation that was clearly associated with HLA-DRB1*0401. Other ExRA manifestations were not associated with individual alleles but with DRB1*04 SE double dose genotypes. This confirms that SE genes contribute to RA disease severity and ExRA. Other genetic and environmental factors may have a more specific impact on individual ExRA manifestations.     

A new species of Achillea (Asteraceae: Anthemideae) from south-east Peloponnisos, Greece

A new species of Achillea L., Achillea occulta Constantin. & Kalpoutz., from the summit area of Mount Koulochera in south-east Peloponnisos, Greece, is described and illustrated. It belongs to A . sect. Ptarmica and further, to a small group of taxa known under the illegitimate sectional name 'Anthemoideae'. In Greece, A. occulta has no close allies. The taxonomically related taxa, i.e. A. barrelieri (Ten.) Sch. Bip., A. mucronulata Bertol., A. oxyloba (DC.) Sch. Bip. and A. schurii Sch. Bip. are found in the Italian Peninsula, the Alps and the Carpathians. Phytogeographically, the finding of a species with such taxonomic connections in south-east Greece was unexpected. The new species grows in semi-shade, often hidden in suitable limestone rock hollows and the foot of rocks, together with several other Greek endemics. Its chromosome number of 2 n  = 18, counted in root tips, is also reported and illustrated.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 249–256.     

A new species of Centaurea (sect. Phalolepis, Compositae: Cardueae) from eastern Peloponnisos, Greece

A new species of Centaurea L. , Centaurea leonidia Kalpoutz. & Constantin. , from two localities west and south-west of the town of Leonidio in eastern Peloponnisos, Greece, is described and illustrated. It belongs to C . sect. Phalolepis , and taxonomically its closest relatives are C. heldreichii Halácsy , a very localized species from south-west Sterea Ellas (Greece) and, surprisingly, C. niederi Heldr., which belongs to sect. Acrolophus. The new species is currently known from two populations of less than 100 individuals each, growing on almost inaccessible cliffs close to the convent of Agios Nikolaos Sintzas (St. Nicolas of Sintza) and the slopes of Poundes summit. Several other Greek endemic species are found in the same areas. Centaurea leonidia is scientifically important as it belongs to a small group of taxa, which, although they are members of sect. Phalolepis , have close allies in sect. Acrolophus. The chromosome number of C. leonidia , 2 n  = 18, counted in root tips, is also reported and illustrated.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 375–383.     

The effects of free-air CO2 enrichment and soil water availability on spatial and seasonal patterns of wheat root growth

Spring wheat [ Triticum aestivum (L). cv. Yecora Rojo] was grown from December 1992 to May 1993 under two atmospheric CO₂ concentrations, 550 μmol mol^–1 for high-CO₂ plots, and 370 μmol mol^–1 for control plots, using a Free-Air CO₂ Enrichment (FACE) apparatus. In addition to the two levels of atmospheric CO₂, there were ample and limiting levels of water supply through a subsurface trip irrigation system in a strip, split-plot design. In order to examine the temporal and spatial root distribution, root cores were extracted at six growth stages during the season at in-row and inter-row positions using a soil core device (86 mm ID, 1.0 m length). Such information would help determine whether and to what extent root morphology is changed by alteration of two important factors, atmospheric CO₂ and soil water, in this agricultural ecosystem. Wheat root growth increased under elevated CO₂ conditions during all observed developmental stages. A maximum of 37% increase in total root dry mass in the FACE vs. Control plots was observed during the period of stem elongation. Greater root growth rates were calculated due to CO₂ enhancement until anthesis. During the early vegetative growth, root dry mass of the inter-row space was significantly higher for FACE than for Control treatments suggesting that elevated CO₂ promoted the production of first-order lateral roots per main axis. Then, during the reproductive period of growth, more branching of lateral roots in the FACE treatment occurred due to water stress. Significant higher root dry mass was measured in the inter-row space of the FACE plots where soil water supply was limiting. These sequential responses in root growth and morphology to elevated CO₂ and reduced soil water supports the hypothesis that plants grown in a high-CO₂ environment may better compensate soil-water-stress conditions.     

Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.     

An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line

An intracellular (ATP + Mg2+)-dependent Ca2+ pumping mechanism has been identified and characterized within the cultured clonal neuroblastoma cell line N1E-115. Using cell suspensions treated with 0.005% saponin which selectively permeabilizes the plasma membrane in 95-98% of the cells, it was possible to show clearly that the intracellular Ca2+ pump mechanism is of non-plasma membrane origin and therefore can be compared directly with the Ca2+ pump characterized in detail in synaptosomal membrane vesicles (Gill, D. L., Grollman, E. F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192; Gill, D. L., Chueh, S. H., and Whitlow, C. L. (1984) J. Biol. Chem. 259, 10807-10813) which was proven by flux reversal studies to be derived from the neural plasma membrane (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990). The intracellular Ca2+ pump in N1E-115 cells is distinct from mitochondrial Ca2+ accumulation and is increased up to 8-fold higher as cells reach confluency. In similarity to the neural plasma membrane pump, the intracellular Ca2+ pump within N1E-115 cells has high affinity for Ca2+ (Km = 0.28 microM), is dependent on both ATP (Km = 26 microM) and either Mg2+ or Mn2+ which half-maximally activate Ca2+ pumping at 0.35 mM and 0.32 mM, respectively, and shows similar specificity for Sr2+ and Ba2+ which half-maximally inhibit Ca2+ transport at 50 microM and 1.5 mM, respectively. In contrast to the neural plasma membrane pump, the intracellular Ca2+ pump displays approximately 40-fold higher sensitivity to La3+ (IC50 = 5 microM) and an apparent 400-fold lower sensitivity to VO4(3-) (IC50 = 185 microM), although the inhibitory effectiveness of VO4(3-) is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO4(3-) in the absence of ATP (apparent IC50 = 5 microM). In further contrast to the neural plasma membrane Ca2+ pump, the intracellular pump within N1E-115 cells is stimulated more than 20-fold by oxalate (giving prolonged linear Ca2+ accumulation), is resistant to low saponin concentrations, and is not modified by calmodulin even after extensive treatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and/or calmodulin antagonist drugs. However, calmidazolium is effective in inhibiting the intracellular Ca2+ pump with an IC50 of approximately 2 microM.     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.