Contrasting strategies of osmotic and ionic regulation in freshwater crabs and shrimps: gene expression of gill ion transporters.

Owing to their extraordinary niche diversity, the Crustacea are ideal for comprehending the evolution of osmoregulation. The processes that effect systemic hydro-electrolytic homeostasis maintain hemolymph ionic composition via membrane transporters located in highly specialized gill ionocytes. We evaluated physiological and molecular hyper- and hypo-osmoregulatory mechanisms in two phylogenetically distant, freshwater crustaceans, the crab Dilocarcinus pagei and the shrimp Macrobrachium jelskii, when osmotically challenged for up to 10 days. When in distilled water, D. pagei survived without mortality, hemolymph osmolality and [Cl-] increased briefly, stabilizing at initial values, while [Na+] decreased continually. Expression of gill V-type H+-ATPase (V-ATPase), Na+/K+-ATPase and Na+/K+/2Cl- symporter genes was unchanged. In M. jelskii, hemolymph osmolality, [Cl-] and [Na+] decreased continually for 12 h, the shrimps surviving only around 15-24 h exposure. Gill transporter gene expression increased 2- to 5-fold. After 10 days exposure to brackish water (25‰S), D. pagei was isosmotic, iso-chloremic and iso-natriuremic. Gill V-ATPase expression decreased while Na+/K+-ATPase and Na+/K+/2Cl- symporter expression was unchanged. In M. jelskii (20‰S), hemolymph was hypo-regulated, particularly [Cl-]. Transporter expression initially increased 3- to 12-fold, declining to control values. Gill V-ATPase expression underlies the ability of D. pagei to survive in fresh water while V-ATPase, Na+/K+-ATPase and Na+/K+/2Cl- symporter expression enables M. jelskii to confront hyper/hypo-osmotic challenges. These findings reveal divergent responses in two unrelated crustaceans inhabiting a similar osmotic niche. While D. pagei does not secrete salt, tolerating elevated cellular isosmoticity, M. jelskii exhibits clear hypo-osmoregulatory ability. Each species has evolved distinct strategies at the transcriptional and systemic levels during its adaptation to fresh water.

hRNAi-mediated knock-down of Sphenophorus levis V-ATPase E in transgenic sugarcane (Saccharum spp interspecific hybrid) affects the insect growth and survival.

KEY MESSAGE: Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.

Whole-exome sequencing reveals novel vacuolar ATPase genes' variants and variants in genes involved in lysosomal biology and autophagosomal formation in oral granular cell tumors.

BACKGROUND: Granular cell tumors (GCTs) are rare neuroectodermal soft tissue neoplasms that mainly affect the skin of the upper limbs and trunks and the oral cavity. GCTs are derived from Schwann cells and, ultrastructurally, their intracytoplasmic granules are considered autophagosomes or autophagolysosomes and are consistent with myelin accumulation. METHODS: In this study, a convenience set of 22 formalin-fixed, paraffin-embedded samples of oral GCTs, all but one sample located at the tongue, was screened for mutations by whole-exome (WES) or targeted sequencing. RESULTS: WES revealed two novel variants in genes of the vacuolar ATPase (V-ATPase) complex: ATP6AP1 frameshift c.746_749del, leading to p.P249Hfs*4, and ATP6V1A non-synonymous c.G868A, leading to p.D290N. Each of these mutations occurred in one case. With regard to the samples that were wild type for these V-ATPase variants, at least two samples presented variants in genes that are part of endosomal/lysosomal/autophagosomal networks including ABCA8, ABCC6, AGAP3, ATG9A, CTSB, DNAJC13, GALC, NPC1, SLC15A3, SLC31A2, and TMEM104. CONCLUSION: Although the mechanisms involved in oral GCT initiation and progression remain unclear, our results suggest that oral GCTs have V-ATPase variants similarly to GCTs from other tissues/organs, and additionally show variants in lysosomes/endosomes/autophagosomal genes.

Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma.

BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.

Immunolocalization of IP3R and V-ATPase in Ameloblastomas.

The goal of this study was to investigate the immunolocalization of inositol 1,4,5-trisphosphate receptor (IP3R) and vacuolar ATPase (V-ATPase) in ameloblastomas with special attention to the invasive front. Thirty-seven cases of previously diagnosed formalin-fixed paraffin-embedded (FFPE) human ameloblastoma samples were selected for this study. The samples were grouped according to the predominant histologic pattern and comprised twelve plexiform, eighteen follicular, and seven unicystic ameloblastomas. Of the unicystic variants, six demonstrated purely luminal and intraluminal growth, and one displayed mural extension. One granular cell variant was included in the follicular ameloblastoma group. All specimens were evaluated for IP3R and V-ATPase expression by immunohistochemistry (IHC). IP3R was positive in columnar cells, similar to ameloblasts, and non-peripheral cells in all samples. In the area of tumor protrusion and front of invasion, membranous and cystoplasmic IP3R expression was observed. In contrast, areas adjacent to tumoral protrusion demonstrated only membranous staining patterns. V-ATPase was not expressed in peripheral columnar cells of the unicystic and granular cell variants of ameloblastoma; however, strong staining was present in these cells in plexiform ameloblastomas, follicular ameloblastomas, and areas of mural growth of unicystic ameloblastomas. In areas of tumor protrusion, reactivity for V-ATPase was observed with both membranous and cytoplasmic staining, while other areas showed only membranous V-ATPase. These findings suggest that concomitant immunolocalization of IP3R and V-ATPase, with both cytoplasmic and membranous expression in the peripheral columnar cells, may indicate the invasive potential of ameloblastomas. Furthermore, these results suggest the tumoral spread of ameloblastomas may be correlated with the autophagy process and channelopathy. The expression of these proteins could establish a baseline for future research and provide therapeutic targets for treatment of ameloblastomas.

Alendronate induces postnatal maxillary bone growth by stimulating intramembranous ossification and preventing premature cartilage mineralization in the midpalatal suture of newborn rats.

Cleft palate is a common malformation of craniofacial development, and postnatal deficiencies in palate formation may occur. The aim of this study was to determine whether alendronate treatment could induce maxillary mineralization and thus reduce the need for surgical procedures. The effects of alendronate on maxillary bone development, the midpalatal suture, and the levels of transforming growth factor beta-1 (TGF-ß1), bone morphogenetic protein 2 (BMP-2), collagen I and II, and V-ATPase were evaluated in newborn rats. Thirty newborn rats were placed in a control group and 30 in a group that received intraperitoneal alendronate (2.5 mg/kg/day). The animals were euthanized on day 7 or 12, and the heads were subjected to histological and immunohistochemical analyses. Specimens from rats that received alendronate presented larger bone matrix deposition in areas of intramembranous ossification of the maxillary bone when compared to controls. Furthermore, higher levels of TGF-ß1, BMP-2, and collagen I were observed, whereas osteoclasts showed no V-ATPase. The alendronate group also showed higher levels of TGF-ß1 and collagen II in the midpalatal suture, whereas BMP-2 levels were lower than in controls. These results coincided with an expansion of the chondroid. In conclusion, alendronate increased the intramembranous ossification in the maxillary bone in association with increased expression of TGF-ß1, BMP-2, and collagen I and decreased V-ATPase. The drug induced an expansion of chondrocytes and a decrease in mineral bone deposition despite the high levels of TGF-ß1 in this area. Alendronate may therefore be useful in the treatment of diseases affecting bone growth.

The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion

Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.