Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.     

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Effect of Temperature on the Fatty Acid Composition of Thermus aquaticus

Thermus aquaticus contains four major fatty acids, iso-C(15) (28%), iso-C(16) (9%), normal-C(16) (13%), and iso-C(17) (48%), when grown at 70 C, as determined by gas chromatography and mass spectrometry. Small amounts of iso-C(12), normal-C(12:1), iso-C(13), normal-C(14), iso-C(14), and normal-C(15:1) were also detected. A change in growth temperature (50 to 75 C at 5-C intervals) affects a shift in the proportions of some of the fatty acids. The proportions of the monoenoic and branched-C(17) fatty acids decreased and the proportions of the higher-melting iso-C(16) and normal-C(16) fatty acids increased. Cells grown at 75 C contained 70% more total fatty acids than cells grown at 50 C. The largest increases, in absolute amounts, were in the content of iso-C(16) and normal-C(16) fatty acids, with only a 1.6-fold increase in the major iso-C(15) and iso-C(17) fatty acids. There was a 2.5-fold decrease in normal-C(15:1) and at least a 24-fold decrease in anteiso-C(17), which is present at 50 and 55 C but not at higher temperatures. There was no difference in proportion or amount of fatty acids between exponential and stationary-phase cells grown at 70 C. When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C(16) and normal-C(16) fatty acids concomitant with a decrease in the proportions of the iso-C(15) and iso-C(17) fatty acids.     

Going, going, gone: the impact of white-nose syndrome on the summer activity of the little brown bat (Myotis lucifugus)

Since its discovery in the winter of 2005-2006, white-nose syndrome (WNS) has killed over one million little brown bats (Myotis lucifugus) in the American northeast. Although many studies have reported die-offs of bats at winter hibernacula, it is important to understand how bat mortality linked to WNS at winter hibernacula affects bat activity levels in their summer ranges. In the summer (May-August) of 2007, 2008 and 2009, we recorded echolocation calls to determine bat activity at sites along the Hudson River, NY (within approx. 100 km of where WNS was first reported). We documented a 78 per cent decline in the summer activity of M. lucifugus, coinciding with the arrival and spread of WNS. We suggest that mortality of M. lucifugus in winter hibernacula is reflected by reduced levels of activity in the summer and that WNS affects the entire bat population of an area, and not only individual hibernacula.     

Genetic architecture,biochemical underpinnings and ecological impact of floral UV patterning

Floral attraction traits can significantly affect pollinator visitation patterns, but adaptive evolution of these traits may be constrained by correlations with other traits. In some cases, molecular pathways contributing to floral attraction are well characterized, offering the opportunity to explore loci potentially underlying variation among individuals. Here, we quantify the range of variation in floral UV patterning (i.e. UV ‘bulls‐eye nectar guides) among crop and wild accessions of Brassica rapa. We then use experimental crosses to examine the genetic architecture, candidate loci and biochemical underpinnings of this patterning as well as phenotypic manipulations to test the ecological impact. We find qualitative variation in UV patterning between wild (commonly lacking UV patterns) and crop (commonly exhibiting UV patterns) accessions. Similar to the majority of crops, recombinant inbred lines (RILs) derived from an oilseed crop × WI fast‐plant^® cross exhibit UV patterns, the size of which varies extensively among genotypes. In RILs, we further observe strong statistical‐genetic and QTL correlations within petal morphological traits and within measurements of petal UV patterning; however, correlations between morphology and UV patterning are weak or nonsignificant, suggesting that UV patterning is regulated and may evolve independently of overall petal size. HPLC analyses reveal a high concentration of sinapoyl glucose in UV‐absorbing petal regions, which, in concert with physical locations of UV‐trait QTLs, suggest a regulatory and structural gene as candidates underlying observed quantitative variation. Finally, insects prefer flowers with UV bulls‐eye patterns over those that lack patterns, validating the importance of UV patterning in pollen‐limited populations of B. rapa.     

Nitrogen and phosphorus accumulation and cycling by Nymphoides peltata (Gmel.) O. Kuntze (Menyanthaceae)

In 1980, the seasonal changes in nitrogen and phosphorus concentration of various plant parts of Nymphoides peltata (Gmel.) O. Kuntze, together with aspects of nitrogen and phosphorus cycling by this species were studied in an oxbow lake of the river Waal (The Netherlands). The nitrogen and phosphorus stores of the water, seston, sediment and macrophyte compartments were assessed each month.The underground Nymphoides structures had high nitrogen and phosphorus concentrations before and after the main growing season, while during summer the aboveground plant parts had high nutrient contents. Nymphoides peltata accumulated maximum amounts of nitrogen (334 mmol m⁻²) and phosphorus (56.6 mmol m⁻²) in July. The upper layers of the bottom appeared to be an enormous nutrient reservoir (94–99% of total) of which the largest part was not directly available to Nymphoides. Nutrient uptake from the sediments by N. peltata is suggested by the fact that the bottom and/or interstitial water of the sample station devoid of rooted macrophytes, contained higher concentrations of nitrogen and phosphorus than that of the Nymphoides stands. The annual flux of nutrients from Nymphoides to the detritus compartment was estimated to be ca. 1200 mmol nitrogen and 164 mmol phosphorus per m² of littoral. During breakdown of the detritus there was a relatively fast net conversion of organically bound nitrogen and phosphorus to inorganic forms, especially at higher temperatures.Nymphoides has the potential to function as an important nitrogen and phosphorus pump, which regenerates sediment nutrients.     

Strikingly high levels of heterozygosity despite 20 years of inbreeding in a clonal honey bee

Inbreeding (the mating between closely related individuals) often has detrimental effects that are associated with loss of heterozygosity at overdominant loci, and the expression of deleterious recessive alleles. However, determining which loci are detrimental when homozygous, and the extent of their phenotypic effects, remains poorly understood. Here, we utilize a unique inbred population of clonal (thelytokous) honey bees, Apis mellifera capensis, to determine which loci reduce individual fitness when homozygous. This asexual population arose from a single worker ancestor approximately 20 years ago and has persisted for at least 100 generations. Thelytokous parthenogenesis results in a 1/3 of loss of heterozygosity with each generation. Yet, this population retains heterozygosity throughout its genome due to selection against homozygotes. Deep sequencing of one bee from each of the three known sub‐lineages of the population revealed that 3,766 of 10,884 genes (34%) have retained heterozygosity across all sub‐lineages, suggesting that these genes have heterozygote advantage. The maintenance of heterozygosity in the same genes and genomic regions in all three sub‐lineages suggests that nearly every chromosome carries genes that show sufficient heterozygote advantage to be selectively detrimental when homozygous.