Ca-ATPase localization and inhibition in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

1985 ◽  
Vol 93 (1-2) ◽  
pp. 157-167 ◽  
Author(s):  
Roni J. Kingsley ◽  
Watabe Norimitsu
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.


2020 ◽  
Author(s):  
Bhairavi Tolani ◽  
Anna Celli ◽  
Yanmin Yao ◽  
Yong Zi Tan ◽  
Richard Fetter ◽  
...  

ABSTRACTMutations in the Ras family of oncogenes are implicated in 33% of human cancers, making Ras an intensely pursued target in drug discovery. As an alternative to direct pharmacological inhibition of Ras, we looked for sensitivities in RAS mutant cells. Using a small molecule screen in cell lines with mutations in Ras and its effector Raf, we discovered 249C as a Ras-mutant selective cytotoxic agent against a spectrum of RAS-mutant cancers. By combining CRISPR chemical-genetic screening, comparative profiling and chemoproteomics, we identified that 249C binds to a unique subunit on vacuolar (V)-ATPase with nanomolar affinity, inhibiting its biochemical activity and, unexpectedly, altering V-ATPase translocation in Ras-induced macropinocytosis. Via binding to V-ATPase, 249C prevents lysosomal acidification and inhibits autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. In characterizing 249C’s mechanism, we show that potency varies with the identity of the RAS driver mutation highlighting a mutant-specific dependence on autophagy and macropinocytosis. Indeed, 249C potently inhibits tumor growth without adverse side effects in a mouse xenograft model of KRAS-driven non-small cell lung cancer. These data establish proof-of-concept for targeting V-ATPase as a way to indirectly target specific Ras mutants, and provide a fundamental link between V-ATPase localization and specific Ras mutant tumor-related activity.


PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8372 ◽  
Author(s):  
Rebecca P. Wenker ◽  
Bradley G. Stevens

Sea whip coral Leptogorgia virgulata are a common structural component of both natural and artificial hard-bottom reef habitats in the mid-Atlantic region and may serve as essential habitat for commercially valuable species. However, they are slow-growing, easily damaged, and especially vulnerable to damage by passive fishing gear such as pots and traps. Despite their potential importance, until recently, sea whips have been generally understudied in this region. We examined the colony complexity, length, age, and growth of sea whips from four artificial reef sites in the mid-Atlantic region to gain a better understanding of their biology in the area. There were no significant differences in the bifurcation (Rb) and tributary to source (T/S) ratios between sites, with the Rb ≈3 for all sites, indicating similar complexity between sites. The total length distribution was 8.3 cm to 85.3 cm, and 50% of corals in the range of 34.2–56.4 cm. Age, estimated from annual growth ring counts, ranged from 2 to 15 y, with 50% of corals in the range of 6 to 8 y. The large proportion of middle-sized and middle-aged corals suggests episodic recruitment. Age-length keys showed the trend of age increasing with total coral length, and a von Bertalanffy growth model demonstrated size-dependent growth following the equation: E[L—t] (cm) = 86.1(1−e−0.14(t−1.44)). This is the first study providing such data for sea whips in the coastal mid-Atlantic region, and the baseline created will be a useful reference to study changes over time.


2011 ◽  
Vol 56 (No. 1) ◽  
pp. 36-48 ◽  
Author(s):  
J. Kuzinski ◽  
R. Zitnan ◽  
T. Viergutz ◽  
J. Legath ◽  
M. Schweigel

In this study we investigated rumen papillae morphology and the localization and expression of the<br />Na<sup>+/</sup>K<sup>+</sup>-ATPase&nbsp;in eight sheep fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c). Four sheep were provided with the ad libitum h-diet for the complete three-week experimental period. The second group of four sheep received the h-diet for only one week and was fed the mixed hay/concentrate (h/c) diet for another two weeks. The amount of concentrate supplement was stepwise increased from 150 to 1000 g/day and given in two meals. Following slaughter rumen papillae from the atrium ruminis (AR), the rumen ventralis (RV) and the ventral blind sac (BSV) were fixed and examined for morphological changes and Na<sup>+</sup>/K<sup>+</sup>-ATPase localization by morphometric methods and immunohistochemistry. Ruminal epithelial cells (REC) originating from the strata basale to granulosum were also isolated. Cellular Na<sup>+</sup>/K<sup>+</sup>-ATPase expression (mRNA and protein) and differentiation state were determined by RT-PCR, Western blot, and flow cytometry. Compared with data from h-fed sheep, morphometric analysis revealed an increased length and width of rumen papillae in h/c-fed sheep, resulting in a marked 41% and 62% increase in rumen papillae surface in AR and RV, respectively. The rumen mucosa of h/c-fed sheep was characterized by a predominant stratum corneum (42 &plusmn; 0.7 &micro;m vs. 28 &plusmn; 0.5 &micro;m), but the thickness of the metabolically active cell layers remained unchanged. REC suspensions from sheep fed the h/c diet generally contained more cells (7.30 &plusmn; 0.83 vs. 3.49 &plusmn; 0.52 &times; 10<sup>7</sup>/ml; P &lt; 0.001) and an increased proportion of REC positive for basal cytokeratin and for the differentiation marker cytokeratin 10 (P &lt; 0.05). Cellular (cell membrane) and epithelial (stratum basale to stratum granulosum) Na<sup>+</sup>/K<sup>+</sup>-ATPase localization was similar between rumen regions and was not changed by concentrate feeding. After two weeks on the h/c-diet, a 96% increase in the absolute number of Na<sup>+</sup>/K<sup>+</sup>-ATPase-positive REC (6.56 &plusmn; 0.84 vs. 3.35 &plusmn; 0.51 &times; 10<sup>7</sup>/ml; P = 0.003) and a 61% elevation (P = 0.043) in Na<sup>+</sup>/K<sup>+</sup>-ATPase protein expression in REC from the upper third of the suprabasal cell layers were found. Moreover, a two-fold (P = 0.001) elevation in cell membrane surface area accompanied by a reduction (1.19 &times; 10<sup>&ndash;7</sup> &plusmn; 1.72 &times; 10<sup>&ndash;9</sup> arbitrary units (AU)/cm2 vs. 1.73 &times; 10<sup>&ndash;7</sup> &plusmn; 8.16 &times; 10<sup>&ndash;9</sup> AU/cm<sup>2</sup> in the h-group; P &lt; 0.001) in specific Na<sup>+</sup>/K<sup>+</sup>-ATPase fluorescence per cm<sup>2</sup> of cell membrane surface area was observed after h/c-feeding. Na<sup>+</sup>/K<sup>+</sup>-ATPase &alpha; subunit mRNA expression was also reduced (P &lt; 0.0001) from 0.154 &plusmn; 0.013 to 0.057 &plusmn; 0.004 pg per pg S18 mRNA control in the h/c-compared with the h-group. Thus, the h/c-diet led to a rapid increase in REC number and total cell membrane surface area in metabolically active and resorptive cell layers and was accompanied by a reduction in Na<sup>+</sup>/K<sup>+</sup>-ATPase mRNA expression and abundance per cell membrane surface area.


1997 ◽  
Vol 200 (20) ◽  
pp. 2653-2662
Author(s):  
J M Lucas ◽  
L W Knapp

The union of calcium cations with carbonate anions to form calcium carbonate (CaCO3) is a fundamentally important physiological process of many marine invertebrates, in particular the corals. In an effort to understand the sources and processes of carbon uptake and subsequent deposition as calcium carbonate, a series of studies of the incorporation of 14C-labeled compounds into spicules was undertaken using the soft coral Leptogorgia virgulata. It has been surmised for some time that dissolved inorganic carbon in sea water is used in the biomineralization process. Furthermore, it was suspected that metabolically generated CO2 is also available for calcification. As a means of testing these possible sources of carbon in spicule calcification, key enzymes or transport systems in each pathway were inhibited. First, the enzyme carbonic anhydrase was specifically inhibited using acetazolamide. Second, the active transport of bicarbonate was inhibited using DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid). Third, CO2 generation resulting from glycolysis and the citric acid cycle was arrested using iodoacetic acid, which interferes specifically with the enzyme glyceraldehyde-3-phosphate dehydrogenase. The results indicate that dissolved CO2 is the largest source of carbon used in the formation of calcitic sclerites, followed by HCO3- from dissolved inorganic carbon. In L. virgulata, the dissolved inorganic carbon is responsible for approximately 67% of the carbon in the sclerites. The other 33% comes from CO2 generated by glycolysis. Two important conclusions can be drawn from this work. First, carbon for spiculogenesis comes not only from dissolved inorganic carbon in the environment but also from metabolically produced carbon dioxide. While the latter has been theorized, it has never before been demonstrated in octocorals. Second, regardless of the carbon source, the enzyme carbonic anhydrase plays a pivotal role in the physiology of spicule formation in Leptogorgia virgulata.


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