Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Plasma membrane H+ pump at a crossroads of acidic and iron stresses in yeast-to-hypha transition.

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

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.

Myrtenal-induced V-ATPase inhibition - A toxicity mechanism behind tumor cell death and suppressed migration and invasion in melanoma.

BACKGROUND: Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice. METHODS: The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach. RESULTS: The inhibition of V-ATPases by 100 µM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4-5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers. CONCLUSIONS: These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition. GENERAL SIGNIFICANCE: The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential.

Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.

BACKGROUND: V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane. METHODS: Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). RESULTS: Cholesterol depletion by 5mM MßCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MßCD. CONCLUSIONS: We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. GENERAL SIGNIFICANCE: The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma.

Vacuolar H+-ATPase in the nuclear membranes regulates nucleo-cytosolic proton gradients.

The regulation of the luminal pH of each organelle is crucial for its function and must be controlled tightly. Nevertheless, it has been assumed that the nuclear pH is regulated by the cytoplasmic proton transporters via the diffusion of H+ across the nuclear pores because of their large diameter. However, it has been demonstrated that ion gradients exist between cytosol and nucleus, suggesting that the permeability of ions across the nuclear pores is restricted. Vacuolar H+-ATPase (V-H+-ATPase) is responsible for the creation and maintenance of trans-membrane electrochemical gradient. We hypothesize that V-H+-ATPase located in the nuclear membranes functions as the primary mechanism to regulate nuclear pH and generate H+ gradients across the nuclear envelope. We studied the subcellular heterogeneity of H+ concentration in the nucleus and cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in prostate cells. Our results indicate that there are proton gradients across the nuclear membranes that are generated by V-H+-ATPase located in the outer and inner nuclear membranes. We demonstrated that these gradients are mostly dissipated by inhibiting V-H+-ATPase. Immunoblots and V-H+-ATPase activity corroborated the existence of V-H+-ATPase in the nuclear membranes. This study demonstrates that V-H+-ATPase is functionally expressed in nuclear membranes and is responsible for nuclear H+ gradients that may promote not only the coupled transport of substrates, but also most electrochemically driven events across the nuclear membranes. This study represents a paradigm shift that the nucleus can regulate its own pH microenvironment, providing new insights into nuclear ion homeostasis and signaling.

A vacuolar H(+)-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress.

BACKGROUND: Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.). METHODS: The vacuolar H(+) pumps, the H(+)-ATPase and H(+)-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops. RESULTS: In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H(+) transport, which was in turn at least 3-folds faster than that mediated by the H(+)-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H(+)-gradient. This effect was antagonized by plant rehydration as the H(+)-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H(+) pumping. Maize tonoplasts exhibited 2-4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H(+) pumping in response to stress relief, resulting in an enhanced H(+)-pumps coupling efficiency. CONCLUSION: These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H(+)-pumps, which differ between weeds and crops upon water deficit. GENERAL SIGNIFICANCE: The evidences highlight an unusual modulation of the H(+)-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.

Polyamines affect the cellular growth and structure of pro-embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines.

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro-embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal-bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H(+) -ATPase P- and V-types and H(+) -PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.

Adição de ácido cítrico potencializa a ação de ácidos húmicos e altera o perfil protéico da membrana plasmática em raízes de milho / Citric acid addition improve humic acids action and change proteins profile from plasma membrane of maize roots

A promoção do crescimento vegetal pelos ácidos húmicos tem sido atribuída a ações similares a hormônios, devido à promoção do desenvolvimento e proliferação das raízes, resultando numa absorção mais eficiente de água e nutrientes. O objetivo deste trabalho foi analisar as mudanças na arquitetura radicular em plântulas de milho e no perfil de proteínas da membrana plasmática (MP) promovidas pelo tratamento com ácidos húmicos (AH) isolados de vermicomposto (20mg C L-1). O efeito da adição de ácido cítrico (AC), importante ácido orgânico presente nos exudados radiculares, sobre a bioatividade destes AH também foi investigada. Foram analisados o comprimento da raiz principal, o número de sítios de mitose, o número e comprimento de raízes laterais e a área radicular total. Para a análise do perfil protéico, vesículas da MP de células de raízes foram obtidas por fracionamento celular e as proteínas analisadas por eletroforese uni (1D) e bidimensional (2D). Observou-se que a adição de AC (0,005mM) aos AH estimularam a promoção do crescimento das raízes laterais (126 por cento), da área radicular (58 por cento) e do número de raízes laterais (55 por cento) em relação às plantas controle. A atividade da bomba de H+ da membrana plasmática, analisada como marcador bioquímico de indução do mecanismo do crescimento ácido, também foi significativamente estimulada (374 por cento) pela solução húmica suplementada com AC. O perfil protéico da MP revelou uma supressão da expressão das proteínas nesta membrana, induzida pelo tratamento com AH e, na presença de AC, esse efeito foi ainda mais evidente. Os resultados obtidos corroboram o mecanismo proposto para a bioatividade de AH no qual a ação de ácidos orgânicos exudados pelas plantas, tais como o AC, promove o rompimento da associação supramolecular dessas substâncias, tornando as moléculas bioativas presentes nos agregados húmicos mais acessíveis aos receptores celulares das raízes.

The plant growth stimulation by humic acids (HA) has been attributed to a hormone-like effect as promoting the root development and proliferation, resulting in a more efficient water and nutrient absorption. This research aims to investigate how the humic acids isolated from vermicompost (20mg L-1) can modify the root architecture and the plasma membrane (PM) protein patterns in maize roots. It was also analyzed the effect of the citric acid (CA), an organic acid present in root exudates. The changes induced in the corn root system were estimated by measuring the taproot length, the amount of root mitotic sites and lateral roots, and the total root area. Plasma membrane vesicles were purified by cell fractionation and the protein patterns were analyzed by uni (1D) and bidimensional (2D) electrophoresis. The results show that the HA in solution with CA (0.005mM) increases the lateral root growth promotion (126 percent), the root area (58 percent), and the number of lateral roots (55 percent). The activity of the plasma membrane H+ pump, analyzed as a marker of the induction of the acid growth mechanism, was also enhanced (374 percent) by the humic solution supplemented with CA. Expression of several plasma membrane proteins was inhibited when plants were treated with HA and this effect was more pronounced upon CA supplementation. The obtained results corroborate the proposed mechanism for the HA bioactivity, by which under the action of root-exuded organic acids, such as CA, a disruption of the HA macrostructure is promoted releasing bioactive molecules presented in the humic aggregates, which becomes more accessible to the root cell receptors.

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H(+) gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H(+)-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H(+)-ATPase (V-ATPase) even in the absence of the H(+) gradient in a water-Me2SO co-solvent mixture, resulting in net synthesis of [γ-(32)P]ATP from [(32)P]Pi and ADP. The water-Me2SO (5 to 20 %) media promoted inhibition of both PPi hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH4 (+)). This effect of Me2SO on the ATP⇔(32)P exchange reaction seems to be related to a decrease of the apparent K m of the V-ATPase for Pi. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.

Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation.

Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H(+ )pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10(-10) and 10(-15) M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H(+)-ATPases and H(+)-pyrophosphatase. Further, the vacuolar H(+)-ATPase had a peak of stimulation in a range from 10(-8) to 10(-10) M IAA, whereas the H(+)-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10(-3) to 10(-15) M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H(+ )pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.

Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots.

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H(+)-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H(+)-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.

Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes.

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.