ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations

The Role of the Polyunsaturated Aldehydes in the Physiology and Ecology of Diatoms

In the last decade diatoms have been shown to release a wide range of secondary metabolites, such as polyunsaturated aldehydes (PUAs), produced by a wound-activated mechanism. PUAs are highly reactive molecules that in most cases induce a drastic reduction in the reproductive response of predators, such as copepods. The variable toxicity of diatoms observed in nature suggests a modulation of PUA production by environmental factors. My results indicate a strong dependence of PUA production on culture age, nutrient limitation and other stress factors, such as UV exposure and low light conditions, in cultures of Skeletonema marinoi, suggesting a direct link between toxin production and cell physiological state. Since it has been hypothesized that PUAs may be released in seawater also as a result of cell mortality, phytoplankton lysis rates were estimated during four oceanographic cruises conducted during periods of diatom blooms in the Northern Adriatic Sea. High lysis rates were observed and preliminary results indicated substantial amounts of PUAs released in seawater. The effects of dissolved PUAs were therefore investigated on organisms other than predators, such as co-occurring bacteria and phytoplankton in culture. PUAs are toxic for several phytoplankton species at concentrations well within the range potentially produced by diatoms. Diatoms can be immunized by sub-lethal concentrations of PUAs. In this case, PUAs may act as signal molecules for bloom termination. PUAs induce also different effects on bacterial growth; some strains are inhibited while others show remarkable resistance to these compounds, and a few of them were even stimulated. Therefore, PUAs appear to have multiple functions in diatoms, namely as chemical defense against grazers, allelochemicals against phytoplankton and bacteria, and signal molecules within their populations

A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.

Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway

Quantification of Dissolved and Particulate Polyunsaturated Aldehydes in the Adriatic Sea

Polyunsaturated aldehydes (PUA) are supposed to play critical roles in chemically-mediated plankton interactions. Laboratory studies suggest that they act as mediators of chemical defense and chemical communication. PUA are oxylipins containing an α,β,γ,δ–unsaturated aldehyde structure element and are mainly found in diatoms. We present here a detailed surface mapping of PUA during a spring bloom of the diatom Skeletonema marinoi in the Adriatic Sea. We monitored dissolved PUA, as well as particulate PUA, which are produced by phytoplankton after cell disintegration. Our survey revealed a patchy distribution of PUA and shows that at most stations S. marinoi is the major contributor to the overall PUA. Our data also suggest that lysis of a diatom bloom can contribute significantly to the dissolved PUA concentrations and that other producers, which are smaller in cell size compared to diatoms, have to be taken into account as well if the total PUA content of marine samples is considered. The analyses of samples collected in deeper water suggests that diatom contribution to PUA decreases with depth, while smaller-sized unidentified organisms take place as dominant contributors to the PUA concentrations

Molecular Basis for Kir6.2 Channel Inhibition by Adenine Nucleotides

AbstractKATP channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the α-phosphate with R201 and interaction of the β-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the β-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the α-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models

Regulation of Cloned Atp–Sensitive K Channels by Phosphorylation, Mgadp, and Phosphatidylinositol Bisphosphate (Pip2): A Study of Channel Rundown and Reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6.2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of KATP channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP2), and phosphorylation. Upon excision of inside-out patches into a Ca2+- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6.2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca2+ accelerated this process, suggesting a role for PIP2 hydrolysis mediated by a Ca2+-dependent phospholipase C. PIP2 could reactivate channel activity after a brief exposure to Ca2+, but not after prolonged exposure. However, in both cases, PIP2 reversed the increase in ATP sensitivity, indicating that PIP2 lowers the ATP sensitivity by increasing Po as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca2+ facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5–15 min, increased ATP sensitivity and loss of reactivation by PIP2 and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP2 responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP2 and phosphorylation

Long Polyamines Act as Cofactors in PIP2 Activation of Inward Rectifier Potassium (Kir2.1) Channels

Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), Gβγ (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody–induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 μM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel–PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels

A Kernel-Based Change Detection Method to Map Shifts in Phytoplankton Communities Measured by Flow Cytometry

1. Automated, ship-board flow cytometers provide high-resolution maps of phytoplankton composition over large swaths of the world\u27s oceans. They therefore pave the way for understanding how environmental conditions shape community structure. Identification of community changes along a cruise transect commonly segments the data into distinct regions. However, existing segmentation methods are generally not applicable to flow cytometry data, as these data are recorded as ‘point cloud’ data, with hundreds or thousands of particles measured during each time interval. Moreover, nonparametric segmentation methods that do not rely on prior knowledge of the number of species are desirable to map community shifts. 2. We present CytoSegmenter, a kernel-based change-point estimation method for segmenting point cloud data. Our method allows us to represent and summarize a point cloud of data points by a single element in a Hilbert space. The change-point locations can be found using a fast dynamic programming algorithm. 3. Through an analysis of 12 cruises, we demonstrate that CytoSegmenter allows us to locate abrupt changes in phytoplankton community structure. We show that the changes in community structure generally coincide with changes in the temperature and salinity of the ocean. We also illustrate how the main parameter of CytoSegmenter can be easily calibrated using limited auxiliary annotated data. 4. CytoSegmenter is generally applicable for segmenting series of point cloud data from any domain. Moreover, it readily scales to thousands of point clouds, each containing thousands of points. In the context of flow cytometry data collected during research cruises, it does not require prior clustering of particles to define taxa labels, eliminating a potential source of error. This represents an important advance in automating the analysis of large datasets now emerging in biological oceanography and other fields. It also allows for the approach to be applied during research cruises

Biological production, export efficiency, and phytoplankton communities across 8000 km of the South Atlantic

Author Posting. © American Geophysical Union, 2017. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 31 (2017): 1066–1088, doi:10.1002/2016GB005488.In situ oxygen tracers (triple oxygen isotope and oxygen/argon ratios) were used to evaluate meridional trends in surface biological production and export efficiency across ~8000 km of the tropical and subtropical South Atlantic in March–May 2013. We used observations of picophytoplankton, nanophytoplankton, and microphytoplankton to evaluate community structure and diversity and assessed the relationships of these characteristics with production, export efficiency, and particulate organic carbon (POC) fluxes. Rates of productivity were relatively uniform along most of the transect with net community production (NCP) between 0 and 10 mmol O2 m−2 d−1, gross primary production (GPP) between 40 and 100 mmol O2 m−2 d−1, and NCP/GPP, a measure of export efficiency, ranging from 0.1 to 0.2 (0.05–0.1 in carbon units). However, notable exceptions to this basin-scale homogeneity included two locations with highly enhanced NCP and export efficiency compared to surrounding regions. Export of POC and particulate nitrogen, derived from sediment traps, correlated with GPP across the transect, over which the surface community was dominated numerically by picophytoplankton. NCP, however, did not correlate with POC flux; the mean difference between NCP and POC flux was similar to published estimates of dissolved organic carbon export from the surface ocean. The interrelated rates of production presented in this work contribute to the understanding, building on the framework of better-studied ocean basins, of how carbon is biologically transported between the atmosphere and the deep ocean.National Science Foundation (NSF) Grant Number: OCE 1029676; Gordon and Betty Moore Foundation Grant Grant Number: 537.01; Woods Hole Oceanographic Institution (WHOI); WHOI Devonshire Postdoctoral Scholarship; National Defense Science and Engineering Graduate Fellowship; WHOI Ocean Life Institute; Woods Hole Oceanographic Institution (WHOI) Ocean and Climate Change Institute NSF Grant Numbers: OCE 1029676, OCE 11543202018-01-1