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PubMed1980

The proteolipid subunit of the chloroplast adenosine triphosphatase complex. Reconstitution and demonstration of proton-conductive properties.

The dicyclohexylcarbodiimide (DCCD)-binding proteolipid of the chloroplast ATPase complex was solubilized in 1-butanol, isolated by ion exchange chromatography, and reconstituted in a liposomal system. Proton-conducting activity was monitored by fluorometry with 9-aminoacridine as an indicator of delta pH in K+-loaded liposomes suspended in a K+-free medium. Addition of valinomycin served to create a membrane potential. Proton mediation was further followed potentiometrically with a pH electrode. Reconstituted chloroplast DCCD-binding proteolipid rapidly catalyzed passive proton movement as measured by the quenching of 9-aminoacridine fluorescence upon addition of valinomycin to K+-loaded vesicles. Proton translocation was inhibited to approximately 80% by hydrophobic dicyclohexylcarbodiimide but not its water-soluble analog (1-ethyl-3[3-dimethylaminopropyl]carbodiimide). Maximal inhibition by DCCD occurred after 60 min of incubation with 20 to 40 nmol of DCCD/20 nmol of proteolipid/10 mumol of lipid. The velocity of proton conduction increased when the external proton concentration in the medium was increased. Similar data were obtained using a pH electrode to follow proton movement. Subjection of proteolipid liposomes to tetranitromethane, resulting in nitration of tyrosine, markedly reduced proton conduction (62% inhibition). In a similar fashion preincubation of reconstituted vesicles with hydrophobic phenylisothiocyanate at neutral pH resulted in a decreased rate of proton movement (50% inhibition) while hydrophilic p-sulfophenylisothiocyanate appeared to have no effect.

Adenosine Triphosphatases↗
PubMed1981

Quantitative aspects of adenosine triphosphate-driven proton translocation in spinach chloroplast thylakoids.

After illumination in the presence of dithiothreitol, chloroplast thylakoids catalyze ATP hydrolysis and an exchange between ATP and Pi in the dark. ATP hydrolysis is linked to inward proton translocation. The relationships between ATP hydrolysis, ATP-Pi exchange, and proton translocation during the steady state were examined. The internal proton concentration was found to be proportional to the rate of ATP hydrolysis when these parameters were varied by procedures that do not alter the proton permeability of the thylakoid membranes. A linear relationship between the internal proton concentration and the rate of nonphosphorylating electron flow was previously verified. By determining the constant relating internal proton concentration to both ATP hydrolysis and electron flow, the proton/ATP ratio for the chloroplast ATPase complex was calculated to be 3.4 +/- 0.3. The presence of Pi, which allows ATP-Pi exchange to occur, lowers the internal proton concentration, but does not alter the relationship between the net rate of ATP hydrolysis and internal proton concentration. ATP-Pi exchange shows a dependence on the proton activity gradient very similar to that of ATP synthesis in the light. These results suggest that ATP-Pi exchange resembles photophosphorylation. In agreement with this idea, it is nucleoside diphosphate from the medium that is phosphorylated during exchange. Moreover, the energy-linked incorporation of Pi and ADP into ATP during exchange occurs at a similar rate. Thus, ATP synthesis from medium ADP and Pi takes place at the expense of the pH gradient generated by ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1996

DCCD-sensitive proton permeability of bacterial photosynthetic membranes. Cross-reconstitution studies with purified bovine heart Fo subunits.

The DCCD-sensitive proton permeability of chromatophores, from a green strain of Rhodobacter Capsulatus is potentiometrically detected following the proton release induced by a transmembrane diffusion potential imposed by a valinomycin-mediated potassium influx with a procedure already used for bovine heart submitochondrial particles (ESMP) and vesicles from Escherichia coli (Zanotti et al. (1994) Eur. J. Biochem. 222, 733-741). In the photosynthetic system, addition of increasing amounts of DCCD inhibits, with a similar titre, both proton permeability and MgATP-dependent ATPase activity as detected in the dark. The titre for 50% inhibition coincides with that obtained measuring proton permeability and ATP hydrolysis in ESMP. Upon removal of F1, the passive proton permeability is much less sensitive to DCCD in chromatophores than in USMP, suggesting that in chromatophores the F1-Fo interaction shapes the DCCD-sensitive proton conducting pathway. Addition of the purified mitochondrial FoI-PVP and oligomycin sensitivity-conferring (OSCP) proteins to the F1 stripped chromatophores restored the sensitivity of proton permeability to DCCD detected in untreated chromatophores. Analysis of the binding of 14C[DCCD] on F1 stripped chromatophores shows that the increase of DCCD sensitivity of proton permeability, caused by addition of mitochondrial Fo proteins, is related to an increase of the binding of the inhibitor to subunit c of Fo sector of ATP synthase complex.

Animals↗
PubMed2004

The influence of subunit III of cytochrome c oxidase on the D pathway, the proton exit pathway and mechanism-based inactivation in subunit I.

Although subunit III of cytochrome c oxidase is part of the catalytic core of the enzyme, its function has remained enigmatic. Comparison of the wild-type oxidase and forms lacking subunit III shows that the presence of subunit III maintains rapid proton uptake into the D pathway at the pH of the bacterial cytoplasm or mitochondrial matrix, apparently by contributing to the protein environment of D132, the initial proton acceptor of the D pathway. Subunit III also appears to contribute to the conformation of the normal proton exit pathway, allowing this pathway to take up protons from the outer surface of the oxidase in the presence of DeltaPsi and DeltapH. Subunit III prevents turnover-induced inactivation of the oxidase (suicide inactivation) and the subsequent loss of Cu(B) from the active site. This function of subunit III appears partly related to its ability to maintain rapid proton flow to the active site, thereby shortening the lifetime of reactive O(2) reduction intermediates. Analysis of proton pumping by subunit III-depleted oxidase forms leads to the proposal that the trapping of two protons in the D pathway, one on E286 and one on D132, is required for efficient proton pumping.

Copper↗
PubMed2001

Patterns of proton pump inhibitor use in clinical practice.

BACKGROUND: Little is known about differences between gastroenterologists and primary care physicians in their patterns of prescribing proton pump inhibitors. SUBJECTS AND METHODS: A survey of practicing primary care physicians from the American Board of Medical Specialties and practicing gastroenterologists from the American Gastroenterological Association was conducted by facsimile. The survey instrument consisted of 13 questions about pharmacokinetics and administration of proton pump inhibitors. RESULTS: The overall response rate was 15% (491 of 3273), and 80% (395 of 491) of respondents were nontrainee gastroenterologists or primary care physicians. Approximately 90% (n = 355) of eligible respondents correctly identified proton pump inhibitors as inhibitors of H+,K+-adenosinetriphosphatase. Proton pump inhibitors were prescribed by 80% (n = 314) of each group for reflux esophagitis. They were prescribed by 67% (122 of 182) of gastroenterologists and 27% (58 of 213) of primary care physicians to prevent ulcers induced by nonsteroidal anti-inflammatory drugs (P <0.001). And they were prescribed by 40% (n = 73) of gastroenterologists and 16% (n = 34) of primary care physicians for uncomplicated heartburn (P <0.001). Proton pump inhibitors were prescribed before a meal by 95% (n = 173) of gastroenterologists and 33% (n = 70) of primary care physicians (P <0.001). Nearly 99% (n = 391) of respondents agreed that proton pump inhibitors were safe, but only 15% (n = 59) thought they should be available without prescription. CONCLUSION: Our survey suggests that the use of proton pump inhibitors differs between gastroenterologists and primary care physicians. Furthermore, although most physicians believe that proton pump inhibitors are safe, few believe that they should be available without a prescription.

Adult↗
PubMed1997

Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; guanidine hydrochloride restores it.

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKa's of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKa's of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKa's of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKa's of E204 and D85.

Bacteriorhodopsins↗
PubMed1999

ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions.

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

Adenosine Diphosphate↗
PubMed2001

A treatment planning comparison of 3D conformal therapy, intensity modulated photon therapy and proton therapy for treatment of advanced head and neck tumours.

BACKGROUND AND PURPOSE: In this work, the potential benefits and limitations of different treatment techniques, based on mixed photon-electron beams, 3D conformal therapy, intensity modulated photons (IM) and protons (passively scattered and spot scanned), have been assessed using comparative treatment planning methods in a cohort of patients presenting with advanced head and neck tumours. MATERIAL AND METHODS: Plans for five patients were computed for all modalities using CT scans to delineate target volume (PTV) and organs at risk (OAR) and to predict dose distributions. The prescribed dose to the PTV was 54 Gy, whilst the spinal cord was constrained to a maximum dose of 40.5 Gy for all techniques. Dose volume histograms were used for physical and biological evaluation, which included equivalent uniform dose (EUD) calculations. RESULTS: Excluding the mixed photon-electron technique, PTV coverage was within the defined limits for all techniques, with protons providing significantly improved dose homogeneity, resulting in correspondingly higher EUD results. For the spinal cord, protons also provided the best sparing with maximum doses as low as 17 Gy. Whilst the IM plans were demonstrated to be significantly superior to non-modulated photon plans, they were found to be inferior to protons for both criteria. A similar result was found for the parotid glands. Although they are partially included in the treated volume there is a clear indication that protons, and to a lesser extent IM photons, could play an important role in preserving organ functionality with a consequent improvement of the patient's quality of life. CONCLUSIONS: For advanced head and neck tumours, we have demonstrated that the use of IM photons or protons both have the potential to reduce the possibility of spinal cord toxicity. In addition, a substantial reduction of dose to the parotid glands through the use of protons enhances the interest for such a treatment modality in cases of advanced head and neck tumours. However, in terms of target coverage, the use of 3D conformal therapy, although somewhat inferior in quality to protons or IM photons, has been shown to be a reasonable alternative to the more advanced techniques. In contrast, the conventional technique of mixed photon and electron fields has been shown to be inferior to all other techniques for both target coverage and OAR involvement.

Carcinoma, Squamous Cell↗
PubMed2002

Proton radiotherapy in management of pediatric base of skull tumors.

PURPOSE: Primary skull base tumors of the developing child are rare and present a formidable challenge to both surgeons and radiation oncologists. Gross total resection with negative margins is rarely achieved, and the risks of functional, structural, and cosmetic deficits limit the radiation dose using conventional radiation techniques. Twenty-nine children and adolescents treated with conformal proton radiotherapy (proton RT) were analyzed to assess treatment efficacy and safety. MATERIALS AND METHODS: Between July 1992 and April 1999, 29 patients with mesenchymal tumors underwent fractionated proton (13 patients) or fractionated combined proton and photon (16 patients) irradiation. The age at treatment ranged from 1 to 19 years (median 12); 14 patients were male and 15 female. Tumors were grouped as malignant or benign. Twenty patients had malignant histologic findings, including chordoma (n = 10), chondrosarcoma (n = 3), rhabdomyosarcoma (n = 4), and other sarcomas (n = 3). Target doses ranged between 50.4 and 78.6 Gy/cobalt Gray equivalent (CGE), delivered at doses of 1.8-2.0 Gy/CGE per fraction. The benign histologic findings included giant cell tumors (n = 6), angiofibromas (n = 2), and chondroblastoma (n = 1). RT doses for this group ranged from 45.0 to 71.8 Gy/CGE. Despite maximal surgical resection, 28 (97%) of 29 patients had gross disease at the time of proton RT. Follow-up after proton RT ranged from 13 to 92 months (mean 40). RESULTS: Of the 20 patients with malignant tumors, 5 (25%) had local failure; 1 patient had failure in the surgical access route and 3 patients developed distant metastases. Seven patients had died of progressive disease at the time of analysis. Local tumor control was maintained in 6 (60%) of 10 patients with chordoma, 3 (100%) of 3 with chondrosarcoma, 4 (100%) of 4 with rhabdomyosarcoma, and 2 (66%) of 3 with other sarcomas. The actuarial 5-year local control and overall survival rate was 72% and 56%, respectively, and the overall survival of the males was significantly superior to that of the female patients (p = 0.002). Of the patients with benign tumors, 1 patient (giant cell tumor) had local failure at 10 months. The other 8 patients continued to have local tumor control; all 9 patients were alive at last follow-up (actuarial 5-year local control and overall survival rate of 89% and 100%, respectively). Severe late effects (motor weakness and sensory deficits) were observed in 2 (7%) of 29 patients. CONCLUSION: Proton RT for children with aggressively recurring tumors after major skull base surgery can offer a considerable prospect of tumor control and survival. Longer follow-up is necessary to assess the real value of protons, in particular with regard to bone growth and cosmetic outcome.

Adolescent↗
PubMed2003

Potential role of intensity-modulated photons and protons in the treatment of the breast and regional nodes.

PURPOSE: To investigate, using comparative treatment planning, the potential improvements that could result through the use of intensity-modulated photons (intensity-modulated radiation therapy [IMRT]) and protons for the locoregional treatment of complex-target breast cancer. METHODS AND MATERIALS: Using CT data from a breast cancer patient, treatment plans were computed using "standard" photon/electron, IMRT, and forward-planned proton techniques. A dose of 50 Gy was prescribed to the target volume consisting of the involved breast, internal mammary, supraclavicular, and axillary nodes. The standard plan was designed using 6-MV X-ray beams to the breast, axillary, and supraclavicular areas and a mixture of 6-MV X-rays and 12-MeV electrons for the internal mammary nodes. Two IMRT (IMX1 and IMX2) plans were calculated for nine evenly spaced beams using dose-volume constraints to the organs at risk. For plan IMX1, precedence was given to optimizing the reduction in lung and heart dose while preserving target dose homogeneity. For plan IMX2, an increased precedence was given to the lungs, heart, and contralateral breast to further reduce doses to these organs and to study the effect on target coverage. The proton plan consisted of two oblique, energy-modulated fields. Target dose homogeneity and the doses to neighboring organs were both considered when comparing the different plans. RESULTS: For the standard plan, dose-volume histograms (DVHs) of the target volumes showed severe dose heterogeneity, whereas target coverage for the IMRT and proton plans was comparable. Lung DVHs for the standard and IMRT plans were also comparable, while the proton plan showed the best sparing over all dose levels. Mean doses to the ipsilateral lung for the three plans were found to be 17 Gy, 15 Gy, and 13 Gy for the standard, IMRT, and proton plans, respectively. For the heart, the IMRT plan delivered the highest mean dose (16 Gy), reflecting the extra dose delivered through this organ to spare the lungs. This was reduced somewhat by the standard plan (15 Gy), with the best sparing being provided by the proton plan (6 Gy). When the IMRT plan was reoptimized with an increased precedence to the normal tissues, the mean doses to all neighboring organs at risk could be reduced, but only at the cost of substantial target dose heterogeneity. CONCLUSIONS: In comparison with the standard plan, IMRT photons have the potential to greatly improve the target dose homogeneity with only a small increase in the doses delivered to the neighboring critical structures. However, when attempting to further reduce doses to the critical structures, substantial loss of target dose homogeneity was found. In conclusion, only the two-field, energy-modulated proton plan had the potential to preserve target dose homogeneity while simultaneously minimizing the dose delivered to both lungs, heart, and the contralateral breast.

Breast Neoplasms↗
PubMed1999

Proton radiation therapy (PRT) for pediatric optic pathway gliomas: comparison with 3D planned conventional photons and a standard photon technique.

PURPOSE: Following adequate therapy, excellent long-term survival rates can be achieved for patients with optic pathway gliomas. Therefore, avoidance of treatment-related functional long-term sequelae is of utmost importance. Optimized sparing of normal tissue is of primary concern in the development of new treatment modalities. The present study compares proton radiation therapy (PRT) with a three-dimensional (3D)-planned multiport photon and a lateral beam photon technique for localized and extensive optic pathway tumors. METHODS AND MATERIALS: Between February 1992 and November 1997, seven children with optic pathway gliomas underwent PRT. For this study, we computed proton, 3D photon, and lateral photon plans based on the same CT data sets, and using the same treatment planning software for all plans. Radiation exposure for normal tissue and discrete organs at risk was quantified based on dose-volume histograms. RESULTS: Gross tumor volume (GTV) ranged from 3.9 cm3 to 127.2 cm3. Conformity index (relation of encompassing isodose to GTV volume) was 2.3 for protons, 2.9 for 3D photons, and 7.3 for lateral photons. The relative increase of normal tissue (NT) encompassed at several isodose levels in relation to NT encompassed by the 95% proton isodose volume was computed. Relative NT volume of proton plan isodoses at the 95%, 90%, 80%, 50%, and 25% isodose level increased from 1 to 1.6, 2.8, 6.4, to a maximum of 13.3. Relative volumes for 3D photons were 1.6, 2.4, 3.8, 11.5, and 34.8. Lateral plan relative values were 6, 8.3, 11.5, 19.2, and 26.8. Analysis for small (<20 cm3) and larger (> 80 cm3) tumors showed that protons encompassed the smallest volumes of NT at all isodose levels. Comparable conformity and high-dose gradient were achieved for proton and 3D photon plans in small tumors. However, with increasing tumor volume and complexity, differences became larger. At the 50% isodose level, 3D photons were superior to lateral photons for small tumors; this advantage was equalized for larger tumors. At the lowest isodose level, 3D photons encompassed the highest amount of NT. Analysis of organs at risk showed that PRT reduced doses to the contralateral optic nerve by 47% and 77% compared to 3D photons and lateral photons, respectively. Reductions were also seen for the chiasm (11% and 16%) and pituitary gland (13% and 16%), with differences at clinically relevant tolerance levels. Furthermore, reduced dose exposure of both temporal lobes (sparing 39% and 54%) and frontal lobes was achieved with PRT. CONCLUSION: PRT offered a high degree of conformity to target volumes and steep dose gradients, thus leading to substantial normal tissue sparing in high- and low-dose areas. It is expected that this will result in decreased long-term toxicity in the maturing child. Advantages of proton versus 3D photon plans became increasingly apparent with increasing target size and tumor complexity. Even in small tumors, conformity of 3D photon irradiation came at the expense of a larger amount of NT receiving moderate to low radiation doses. Lateral photons resulted in inferior dose distribution with high radiation exposure of clinically relevant normal tissues.

Child↗
PubMed1996

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.

Carbon Monoxide↗
PubMed2000

Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin.

The transport of protons across membranes is an important process in cellular bioenergetics. The light-driven proton pump bacteriorhodopsin is the best-characterized protein providing this function. Photon energy is absorbed by the chromophore retinal, covalently bound to Lys 216 via a protonated Schiff base. The light-induced all-trans to 13-cis isomerization of the retinal results in deprotonation of the Schiff base followed by alterations in protonatable groups within bacteriorhodopsin. The changed force field induces changes, even in the tertiary structure, which are necessary for proton pumping. The recent report of a high-resolution X-ray crystal structure for the late M intermediate of a mutant bacteriorhopsin (with Asp 96-->Asn) displays the structure of a proton pathway highly disturbed by the mutation. To observe an unperturbed proton pathway, we determined the structure of the late M intermediate of wild-type bacteriorhodopsin (2.25 A resolution). The cytoplasmic side of our M2 structure shows a water net that allows proton transfer from the proton donor group Asp 96 towards the Schiff base. An enlarged cavity system above Asp 96 is observed, which facilitates the de- and reprotonation of this group by fluctuating water molecules in the last part of the cycle.

Bacteriorhodopsins↗
PubMed2003

The requirement of cytosolic phospholipase A2 for the PMA activation of proton efflux through the N-terminal 230-amino-acid fragment of gp91phox.

The absolute requirement for the 85 kDa cytosolic phospholipase A(2) (cPLA(2)) in the PMA stimulation of proton efflux through the NADPH-oxidase-associated proton channel, has previously been demonstrated using a PLB-985 cell line deficient in cPLA(2) (PLB-D). The flux of protons in Chinese-Hamster ovary (CHO) cells that express the N-terminal 230-amino-acid (NT) fragment of gp91(phox) is activated by arachidonic acid (AA) added externally. To investigate the physiological role of cPLA(2), and the intracellular AA that it releases, in the activation of proton flux through the NT fragment of gp91(phox), this fragment was stably expressed in PLB-985 cells (PLB-985 NT) and in PLB-D cells (PLB-D NT). The expression of the NT fragment of gp91(phox) by itself in PLB-985 did not initiate differentiation and did not alter their ability to undergo differentiation after the addition of DMSO. Addition of PMA induced a proton efflux from undifferentiated PLB-985 NT cells expressing the NT fragment of gp91(phox), which was inhibited by zinc. In contrast, PMA failed to activate proton efflux in undifferentiated PLB-D NT cells, lacking the expression of cPLA(2); however, addition of AA restored the efflux of protons in these cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in the activation of proton flux through the NT fragment of gp91(phox).

Cadmium↗
PubMed2000

Inactivation of human normal and tumour cells irradiated with low energy protons.

PURPOSE: To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation. MATERIALS AND METHODS: Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed. RESULTS: Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively. CONCLUSIONS: RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

Cell Line↗
PubMed2003

Intensity modulated radiation therapy using laser-accelerated protons: a Monte Carlo dosimetric study.

In this paper we present Monte Carlo studies of intensity modulated radiation therapy using laser-accelerated proton beams. Laser-accelerated protons coming out of a solid high-density target have broad energy and angular spectra leading to dose distributions that cannot be directly used for therapeutic applications. Through the introduction of a spectrometer-like particle selection system that delivers small pencil beams of protons with desired energy spectra it is feasible to use laser-accelerated protons for intensity modulated radiotherapy. The method presented in this paper is a three-dimensional modulation in which the proton energy spectrum and intensity of each individual beamlet are modulated to yield a homogeneous dose in both the longitudinal and lateral directions. As an evaluation of the efficacy of this method, it has been applied to two prostate cases using a variety of beam arrangements. We have performed a comparison study between intensity modulated photon plans and those for laser-accelerated protons. For identical beam arrangements and the same optimization parameters, proton plans exhibit superior coverage of the target and sparing of neighbouring critical structures. Dose-volume histogram analysis of the resulting dose distributions shows up to 50% reduction of dose to the critical structures. As the number of fields is decreased, the proton modality exhibits a better preservation of the optimization requirements on the target and critical structures. It is shown that for a two-beam arrangement (parallel-opposed) it is possible to achieve both superior target coverage with 5% dose inhomogeneity within the target and excellent sparing of surrounding tissue.

Algorithms↗
PubMed1985

Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.

Ammonia↗
PubMed2002

Modulation of proton pumping across proteoliposome membranes reconstituted with tonoplast H(+)-ATPase from cultured rice (Oryza sativa L. var. Boro) cells by acyl steryl glucoside and steryl glucoside.

Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.

Biological Transport↗