Inhibition of vascular smooth muscle cell calcification by ATP analogues

Purinergic Signalling - Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like....     

Biogeographic effects of red fire ant invasion

The red imported fire ant, Solenopsis invicta , was accidentally introduced to North America over 60 years ago and has spread throughout the southeastern United States. We document the biogeographic consequences of this invasion. We censused ground-foraging ant communities on a 2000 km transect from Florida through New York that passed through invaded and intact biotas. Native ant species density peaks at mid-latitudes in the eastern United States, and the location of this peak corresponds to the range limit of S. invicta . In uninvaded sites, ant species co-occur less often than expected by chance. In the presence of S. invicta , community structure converges to a random pattern. Our results suggest that the effects of S. invicta on native ant communities are pervasive: not only does the presence of S. invicta reduce species density at local scales, it alters the co-occurrence patterns of surviving species at a biogeographic scale.     

Acidosis, hypoxia and bone

Bone homeostasis is profoundly affected by local pH and oxygen tension. It has long been recognised that the skeleton contains a large reserve of alkaline mineral (hydroxyapatite), which is ultimately available to neutralise metabolic H⁺ if acid-base balance is not maintained within narrow limits. Bone cells are extremely sensitive to the direct effects of pH: acidosis inhibits mineral deposition by osteoblasts but it activates osteoclasts to resorb bone and other mineralised tissues. These reciprocal responses act to maximise the availability of OH⁻ ions from hydroxyapatite in solution, where they can buffer excess H⁺. The mechanisms by which bone cells sense small pH changes are likely to be complex, involving ion channels and receptors in the cell membrane, as well as direct intracellular effects. The importance of oxygen tension in the skeleton has also long been known. Recent work shows that hypoxia blocks the growth and differentiation of osteoblasts (and thus bone formation), whilst strongly stimulating osteoclast formation (and thus bone resorption). Surprisingly, the resorptive function of osteoclasts is unimpaired in hypoxia. In vivo, tissue hypoxia is usually accompanied by acidosis due to reduced vascular perfusion and increased glycolytic metabolism. Thus, disruption of the blood supply can engender a multiple negative impact on bone via the direct actions of reduced pO₂ and pH on bone cells. These observations may contribute to our understanding of the bone disturbances that occur in numerous settings, including ageing, inflammation, fractures, tumours, anaemias, kidney disease, diabetes, respiratory disease and smoking.     

No phospholipid monolayer-sugar interactions

Studies by a number of workers using the Langmuir film balance have shown that when carbohydrates, such as sucrose or glycerol, are dissolved in a subphase on which a phospholipid is spread, film expansion occurs (Cadenhead & Demchak, 1969; Cadenhead & Bean, 1972; Maggio et al., 1976; Maggio & Lucy, 1978). Recently such effects have been observed again, particularly with the carbohydrates galactose and trehalose (Johnston et al., 1984). The origin of these film expansions was uncertain, and various suggestions have been made to explain them. One idea was that they might be due to interactions which these carbohydrates have with the water molecules close to the polar head groups of the lipids. Recent studies in our two laboratories, described here, show that the magnitude of the expansion effects is variable and that in general they arise from surfactant impurities in the sugars. These impurities are observed in carbohydrates which are reputedly of high grade; the amount of impurity present can vary from batch to batch, and sometimes they can be difficult to remove. Film balance techniques or subphase preparation can mask the detection of minor impurities. The presence of surfactant impurities in reputedly pure carbohydrates needs to be considered in other biochemical and biophysical studies of lipids and cell membranes.     

Genome-wide linkage scans for loci affecting total cholesterol, HDL-C, and triglycerides: the Family Blood Pressure Program

Atherosclerosis accounts for 75% of all deaths from cardiovascular disease and includes coronary heart disease (CHD), stroke, and other diseases of the arteries. More than half of all CHD is attributable to abnormalities in levels and metabolism of lipids. To locate genes that affect total cholesterol, high density lipoprotein cholesterol (HDL-C), and triglycerides, genome-wide linkage scans for quantitative trait loci were performed using variance components methods as implemented in SOLAR on a large diverse sample recruited as part of the Family Blood Pressure Program. Phenotype and genetic marker data were available for 9,299 subjects in 2,953 families for total cholesterol, 8,668 subjects in 2,736 families for HDL, and 7,760 subjects in 2,499 families for triglycerides. Mean lipid levels were adjusted for the effects of sex, age, age², age-by-sex interaction, body mass index, smoking status, and field center. HDL-C and triglycerides were further adjusted for average total alcoholic drinks per week and estrogen use. Significant linkage was found for total cholesterol on chromosome 2 (LOD = 3.1 at 43 cM) in Hispanics and for HDL-C on chromosome 3 (LOD = 3.0 at 182 cM) and 12 (LOD = 3.5 at 124 cM) in Asians. In addition, there were 13 regions that showed suggestive linkage (LOD ≥ 2.0); 7 for total cholesterol, 4 for HDL, and 2 for triglycerides. The identification of these loci affecting lipid phenotypes and the apparent congruence with previous linkage results provides increased support that these regions contain genes influencing lipid levels.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Hypoxia stimulates vesicular ATP release from rat osteoblasts

Many neuronal and non‐neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y₁ receptor, stimulate osteoclast formation and activity, whilst P2Y₂ receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation‐dependent manner, with mature, bone‐forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N‐ethylmaleimide (1–1,000 µM) inhibited basal ATP release by up to 99%. The presence of granular ATP‐filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O₂) for up to 3 min increased ATP release from osteoblasts up to 2.5‐fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 µM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N‐ethylmaleimide (100 µM) attenuated the hypoxia‐induced ATP release by up to 80%. Depletion of quinacrine‐stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O₂ could influence local purinergic signaling and affect the balance between bone formation and bone resorption. J. Cell. Physiol. 220: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis

It has long been known that core body temperature declines with age, with temperatures of 35.5°C or below common in the elderly. However, the effects of temperature reduction on bone cell function and skeletal homeostasis have been little studied. We investigated the effects of mild hypothermia (35.5°C) and severe hypothermia (34°C) on bone-forming osteoblasts, and bone-resorbing osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by 75% at 35.5°C and by 95% at 34°C after 14-16 days culture, compared to 37°C. In addition to reductions in osteoblast cell number, expression of mRNAs for Runx2, alkaline phosphatase, osteocalcin and type I collagen were also down-regulated in hypothermia. In contrast, formation of osteoclasts in mononuclear cell cultures derived from mouse marrow, showed a 1.5 to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Taken together, these data show that hypothermia exerts reciprocal effects on bone cell function by retarding osteoblast differentiation and bone formation, whilst increasing osteoclastogenesis and thus resorption. These results suggest the possibility that hypothermia in the elderly could potentially have a direct, negative impact on bone metabolism.     

A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity,but Helicase Activity Is Dispensable

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase α-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.