Inhibition of calcium-independent phospholipase A impairs agonist-induced calcium entry in keratinocytes

BACKGROUND: In many cells, depletion of intracellular calcium (Ca2+) reservoirs triggers Ca2+ entry through store-operated Ca2+ channels in the plasma membrane. However, the mechanisms of agonist-induced calcium entry (ACE) in keratinocytes are not fully understood. OBJECTIVES: This study was designed to determine if pharmacological inhibition of calcium-independent phospholipase A (iPLA(2)) impairs ACE in normal human epidermal keratinocytes. METHODS: Confocal laser scanning microscopy was used to monitor the dynamics of Ca2+ signalling in keratinocytes loaded with the calcium-sensitive dye Fluo-4. Cells were stimulated with extracellular nucleotides [adenosine triphosphate (ATP) or uridine triphosphate (UTP)] or with lysophosphatidic acid (LPA), a bioactive lipid that regulates keratinocyte proliferation and differentiation. RESULTS: Both ATP and UTP induced Ca2+ release in primary human keratinocytes. This was not followed by robust Ca2+ influx when the experiments were performed in low Ca2+ (70 micromol L(-1)) medium. Upon elevation of extracellular Ca2+ to 1.2 mmol L(-1), however, a biphasic response consisting of an initial Ca2+ peak followed by an elevated plateau was observed. The plateau phase was inhibited when cells were treated with bromoenol lactone, a specific pharmacological inhibitor of iPLA(2). These findings indicate that iPLA(2) activity is required for ACE in keratinocytes. LPA also evoked Ca2+ release in keratinocytes but failed to induce sustained Ca2+ entry even when extracellular Ca2+ was elevated to 1.2 mmol L(-1). CONCLUSION: Our results demonstrate for the first time an important role for iPLA(2) in regulating ACE in primary human keratinocytes

Activation of store-operated calcium entry in airway smooth muscle cells: insight from a mathematical model

Intracellular dynamics of airway smooth muscle cells (ASMC) mediate ASMC contraction and proliferation, and thus play a key role in airway hyper-responsiveness (AHR) and remodelling in asthma. We evaluate the importance of store-operated entry (SOCE) in these dynamics by constructing a mathematical model of ASMC signaling based on experimental data from lung slices. The model confirms that SOCE is elicited upon sufficient depletion of the sarcoplasmic reticulum (SR), while receptor-operated entry (ROCE) is inhibited in such conditions. It also shows that SOCE can sustain agonist-induced oscillations in the absence of other influx. SOCE up-regulation may thus contribute to AHR by increasing the oscillation frequency that in turn regulates ASMC contraction. The model also provides an explanation for the failure of the SERCA pump blocker CPA to clamp the cytosolic of ASMC in lung slices, by showing that CPA is unable to maintain the SR empty of . This prediction is confirmed by experimental data from mouse lung slices, and strongly suggests that CPA only partially inhibits SERCA in ASMC

Osmosensitivity of Transient Receptor Potential Vanilloid 1 Is Synergistically Enhanced by Distinct Activating Stimuli Such as Temperature and Protons

In animals, body-fluid osmolality is continuously monitored to keep it within a narrow range around a set point (∼300 mOsm/kg). Transient receptor potential vanilloid 1 (TRPV1), a cation channel, has been implicated in body-fluid homeostasis in vivo based on studies with the TRPV1-knockout mouse. However, the response of TRPV1 to hypertonic stimuli has not been demonstrated with heterologous expression systems so far, despite intense efforts by several groups. Thus, the molecular entity of the hypertonic sensor in vivo still remains controversial. Here we found that the full-length form of TRPV1 is sensitive to an osmotic increase exclusively at around body temperature using HEK293 cells stably expressing rat TRPV1. At an ambient temperature of 24°C, a slight increase in the intracellular calcium concentration ([Ca2+]i) was rarely observed in response to hypertonic stimuli. However, the magnitude of the osmosensitive response markedly increased with temperature, peaking at around 36°C. Importantly, the response at 36°C showed a robust increase over a hypertonic range, but a small decrease over a hypotonic range. A TRPV1 antagonist, capsazepine, and a nonspecific TRP channel inhibitor, ruthenium red, completely blocked the increase in [Ca2+]i. These results endorse the view that the full-length form of TRPV1 is able to function as a sensor of hypertonic stimuli in vivo. Furthermore, we found that protons and capsaicin likewise synergistically potentiated the response of TRPV1 to hypertonic stimuli. Of note, HgCl2, which blocks aquaporins and inhibits cell-volume changes, significantly reduced the osmosensitive response. Our findings thus indicate that TRPV1 integrates multiple different types of activating stimuli, and that TRPV1 is sensitive to hypertonic stimuli under physiologically relevant conditions

The Winchcombe meteorite, a unique and pristine witness from the outer solar system.

Direct links between carbonaceous chondrites and their parent bodies in the solar system are rare. The Winchcombe meteorite is the most accurately recorded carbonaceous chondrite fall. Its pre-atmospheric orbit and cosmic-ray exposure age confirm that it arrived on Earth shortly after ejection from a primitive asteroid. Recovered only hours after falling, the composition of the Winchcombe meteorite is largely unmodified by the terrestrial environment. It contains abundant hydrated silicates formed during fluid-rock reactions, and carbon- and nitrogen-bearing organic matter including soluble protein amino acids. The near-pristine hydrogen isotopic composition of the Winchcombe meteorite is comparable to the terrestrial hydrosphere, providing further evidence that volatile-rich carbonaceous asteroids played an important role in the origin of Earth's water

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment

Background: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. Results: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. Conclusion: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas

Calcium mobilization via intracellular ion channels, store organization and mitochondria in smooth muscle

In smooth muscle, Ca2+ release from the internal store into the cytoplasm occurs via inositol trisphosphate (IP3R) and ryanodine receptors (RyR). The internal Ca2+ stores containing IP3R and RyR may be arranged as multiple separate compartments with various IP3R and RyR arrangements, or there may be a single structure containing both receptors. The existence of multiple stores is proposed to explain several physiological responses which include the progression of Ca2+ waves, graded Ca2+ release from the store and various local responses and sensitivities. We suggest that, rather than multiple stores, a single luminally-continuous store exists in which Ca2+ is in free diffusional equilibrium throughout. Regulation of Ca2+ release via IP3R and RyR by the local Ca2+ concentration within the stores explains the apparent existence of multiple stores and physiological processes such as graded Ca2+ release and Ca2+ waves. Close positioning of IP3R on the store with mitochondria or with receptors on the plasma membrane creates ‘IP3 junctions’ to generate local responses on the luminally-continuous store

Valorisation of Biowastes for the Production of Green Materials Using Chemical Methods

With crude oil reserves dwindling, the hunt for a sustainable alternative feedstock for fuels and materials for our society continues to expand. The biorefinery concept has enjoyed both a surge in popularity and also vocal opposition to the idea of diverting food-grade land and crops for this purpose. The idea of using the inevitable wastes arising from biomass processing, particularly farming and food production, is, therefore, gaining more attention as the feedstock for the biorefinery. For the three main components of biomass—carbohydrates, lipids, and proteins—there are long-established processes for using some of these by-products. However, the recent advances in chemical technologies are expanding both the feedstocks available for processing and the products that be obtained. Herein, this review presents some of the more recent developments in processing these molecules for green materials, as well as case studies that bring these technologies and materials together into final products for applied usage