Active transport of proton and calcium in higher plant cells.

Membrane transport of proton and calcium (Ca2+) plays a fundamental role in growth and developmental processes in higher plant cells. The plasma membrane contains an ATPase (P-ATPase) that pumps protons into the extracellular space, whereas two proton pumps, a vacuolar-type ATPase (V-ATPase) and a pyrophosphatase (H+-PPase) are associated with the tonoplast and pump protons into the vacuole. The P-ATPase, V-ATPase and H+-PPase catalyse electrogenic H+-translocation, giving rise to a proton motive force used to transport different molecules, via specific transport proteins (channels or carriers: H+-symport or H+-antiport), across the plasma membrane and the tonoplast

Purification and functional reconstitution of the tonoplast pyrophosphate-dependent proton pump fron Rubus hispidus cell cultures.

The hydrolytic subunit of the H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1.) prepared from Rubus hispidus cell cultures has been purified from tonoplast-enriched membranes and analysed by SDS-polyacrylamide gel electrophoresis, Only one polypeptide of M(r) 70 000 was recovered with the V-PPase activity after solubilization in the presence of Triton X-100, purification by gel filtration (Superose) and anion exchange (Mono Q) chromatography. This polypeptide strongly cross-reacted with an antibody raised against the V-PPase from Vigna radiata. The tonoplast-enriched fraction was also used to solubilize and reconstitute the-V-PPase. The proteoliposomes showing a PPi-dependent proton transport activity were purified by gel filtration (Superose) and analysed by SDS-polyacrylamide gel electrophoresis. Only one polypeptide of M(r) 70 000 was recovered with the proton-pumping activity. All these data suggest that the native V-PPase from Rubus is composed of a single kind of polypeptide with an M(r) of 70 000 and representing the catalytic subunit.

Trans-scleral local resection of toxic choroidal melanoma after proton beam radiotherapy.

AIM: To report on trans-scleral local resection of choroidal melanoma for exudative retinal detachment and neovascular glaucoma (toxic tumour syndrome) after proton beam radiotherapy (PBR). METHODS: A non-randomised, prospective study of secondary trans-scleral local resection of choroidal melanoma for exudative retinal detachment with or without neovascular glaucoma after PBR. The patients were treated at the Liverpool Ocular Oncology Centre between February 2000 and April 2008. The trans-scleral local resection was performed with a lamellar-scleral flap, using systemic hypotension to reduce haemorrhage. RESULTS: 12 patients (six women, six men) with a mean age of 51 years (range 20-75) were included in this study. The tumour margins extended anterior to ora serrata in six patients. On ultrasonography, the largest basal tumour dimension averaged 12.4 mm (range 6.8-18.1) and the tumour height averaged 7.1 mm (range 4.2-10.7). The retinal detachment was total in seven patients. Neovascular glaucoma was present in four patients. The time between PBR and local resection had a mean of 17.4 months (range 1-84). The ophthalmic follow-up time after the local resection had a mean of 46.2 months (range 14-99). At the latest known status, the eye was conserved in 10 patients, with a flat retina in all these patients and visual acuity equal or better than 6/30 in four patients. The reasons for enucleation were: patient request for enucleation when rhegmatogenous retinal detachment complicated the resection (one patient) and phthisis (one patient). CONCLUSIONS: Exudative retinal detachment, rubeosis and neovascular glaucoma after PBR of a choroidal melanoma can resolve after trans-scleral local resection of the tumour. Our findings suggest that these complications are caused by the persistence of the irradiated tumour within the eye ('toxic tumour syndrome').

In vivo metabolic profiling of glioma-initiating cells using proton magnetic resonance spectroscopy at 14.1 Tesla.

In the last decade, evidence has emerged indicating that the growth of a vast majority of tumors including gliomas is sustained by a subpopulation of cancer cells with stem cell properties called cancer initiating cells. These cells are able to initiate and propagate tumors and constitute only a fraction of all tumor cells. In the present study, we showed that intracerebral injection of cultured glioma-initiating cells into nude mice produced fast growing tumors showing necrosis and gadolinium enhancement in MR images, whereas gliomas produced by injecting freshly purified glioma-initiating cells grew slowly and showed no necrosis and very little gadolinium enhancement. Using proton localized spectroscopy at 14.1 Tesla, decreasing trends of N-acetylaspartate, glutamate and glucose concentrations and an increasing trend of glycine concentration were observed near the injection site after injecting cultured glioma-initiating cells. In contrast to the spectra of tumors grown from fresh cells, those from cultured cells showed intense peaks of lipids, increased absolute concentrations of glycine and choline-containing compounds, and decreased concentrations of glutamine, taurine and total creatine, when compared with a contralateral non-tumor-bearing brain tissue. A decrease in concentrations of N-acetylaspartate and γ-aminobutyrate was found in both tumor phenotypes after solid tumor formation. Further investigation is needed to determine the cause of the dissimilarities between the tumors grown from cultured glioma-initiating cells and those from freshly purified glioma-initiating cells, both derived from human glioblastomas.

Transpalpebral proton beam radiotherapy of choroidal melanoma.

BACKGROUND: Collateral damage to upper eyelid margin during proton beam radiotherapy (PBR) for choroidal melanoma may cause squamous metaplasia of the tarsal conjunctiva with keratinisation, corneal irritation, discomfort and, rarely, corneal perforation. We evaluated transpalpebral PBR as a means of avoiding collateral damage to the upper eyelid margin without increasing the risk of failure of local tumour control. METHODS: Retrospective study of consecutive patients who underwent PBR for choroidal melanoma between 1992 and 2007 at the Royal Liverpool University Hospital and the Douglas Cyclotron at Clatterbridge Cancer Centre, UK. RESULTS: Sixty-three patients were included in this study. Mean basal tumour diameter and tumour thickness were 11.8 mm and 3.6 mm, respectively. PBR mean beam range and modulation were 26.5 mm and 16.9 mm respectively. The eyelid margin was included in the radiation field in 15 (24%) eyes. The median follow-up was 2.5 years. Local tumour recurrence developed in 2 (3.2%) patients. In these two cases that developed tumour recurrence the transpalpebral treatment did not involve the eyelid margin. Six (9.5%) patients died of metastatic disease. No eyelid or ocular surface problems developed in any of the 48 patients who were treated without eyelid rim involvement, while 7 of the 15 patients with unavoidable irradiation of the eyelid rim developed some degree of madarosis. These seven patients all received more than 26.55 proton Gy to the eyelid margin. Symptoms, such as grittiness occurred in 12% of 48 patients without eyelid margin irradiation as compared with 53% of 15 patients whose lid margin was irradiated. CONCLUSIONS: Transpalpebral PBR of choroidal melanoma avoids eyelid and ocular surface complications without increasing failure of local tumour control.

Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes.

A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl-transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH-cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes.

Role of vitreoretinal surgery in maximizing treatment outcome following complications after proton therapy for uveal melanoma.

L'utilisation de faisceaux de protons accélérés dans le traitement des mélanomes de l'uvée a été utilisée pour la première fois en Suisse (et par ailleurs en Europe) en 1984. Depuis, la protonthérapie a constamment évolué avec des logiciels toujours plus performants et précis pour devenir à l'heure actuelle le traitement de référence pour ce type de tumeurs. Ainsi, jusqu'à ce jour, l'Institut Paul Scherrer à Villigen a traité plus de 7000 cas de tumeurs oculaires. Mais la protonthérapie, aussi efficace soit-elle avec un taux de guérison de plus de 98%, comporte malheureusement un certain nom bre d'effets secondaires et indésirables pouvant parfois mener le patient jusqu'à l'énucléation secondai re. De la simple dermatite actinique à l'hémorragie intravitréenne massive, les complications induites sont pour la plupart bien connues et documentées mais leurs prises en charge, notamment sur un organe préalablement irradié diffèrent. Alors que nous avons beaucoup de recul sur la protonthérapie, la gestion de ses complications reste propre à chaque centre de soin et n'est que très peu documentée. Les complications majeures de la protonthérapie qui ont nécessité une prise en charge par le chirurgien vitrorétinien représentent souvent un défi majeur. Bien que rares, puisqu'elles ne représentent que 2% de notre collectif, celles-ci peuvent avoir de lourdes conséquences. Pa r exemple, une hémorragie intravitréenne massive, complication la plus fréquente dans notre série, compromet l'observation de la tumeur au fond d'oeil et empêche le bon suivi oncologique. La chirurgie vitrorétinienne a alors pour mission, de restaurer la transparence des milieux, élément indispensable à l'ophtalmologue pour le suivi clinique, iconographique et radiologique des mélanomes de l'uvée. Secondairement, cette chirurgie permet parfois d'augmenter l'acuité visuelle de l'oeil malade. La chirurgie vitrorétinienne est un précieux atout pour l'oncologue et permet d'éviter une énucléation secondaire. Elle participe ainsi à la prise en charge globale du patient atteint de mélanome de l'uvée.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Clinical Proton MR Spectroscopy in Central Nervous System Disorders.

A large body of published work shows that proton (hydrogen 1 [(1)H]) magnetic resonance (MR) spectroscopy has evolved from a research tool into a clinical neuroimaging modality. Herein, the authors present a summary of brain disorders in which MR spectroscopy has an impact on patient management, together with a critical consideration of common data acquisition and processing procedures. The article documents the impact of (1)H MR spectroscopy in the clinical evaluation of disorders of the central nervous system. The clinical usefulness of (1)H MR spectroscopy has been established for brain neoplasms, neonatal and pediatric disorders (hypoxia-ischemia, inherited metabolic diseases, and traumatic brain injury), demyelinating disorders, and infectious brain lesions. The growing list of disorders for which (1)H MR spectroscopy may contribute to patient management extends to neurodegenerative diseases, epilepsy, and stroke. To facilitate expanded clinical acceptance and standardization of MR spectroscopy methodology, guidelines are provided for data acquisition and analysis, quality assessment, and interpretation. Finally, the authors offer recommendations to expedite the use of robust MR spectroscopy methodology in the clinical setting, including incorporation of technical advances on clinical units. © RSNA, 2014 Online supplemental material is available for this article.

Proton T1 relaxation times of metabolites in human occipital white and gray matter at 7 T.

Proton T1 relaxation times of metabolites in the human brain have not previously been published at 7 T. In this study, T1 values of CH3 and CH2 group of N-acetylaspartate and total creatine as well as nine other brain metabolites were measured in occipital white matter and gray matter at 7 T using an inversion-recovery technique combined with a newly implemented semi-adiabatic spin-echo full-intensity acquired localized spectroscopy sequence (echo time = 12 ms). The mean T1 values of metabolites in occipital white matter and gray matter ranged from 0.9 to 2.2 s. Among them, the T1 of glutathione, scyllo-inositol, taurine, phosphorylethanolamine, and N-acetylaspartylglutamate were determined for the first time in the human brain. Significant differences in T1 between white matter and gray matter were found for water (-28%), total choline (-14%), N-acetylaspartylglutamate (-29%), N-acetylaspartate (+4%), and glutamate (+8%). An increasing trend in T1 was observed when compared with previously reported values of N-acetylaspartate (CH3 ), total creatine (CH3 ), and total choline at 3 T. However, for N-acetylaspartate (CH3 ), total creatine, and total choline, no substantial differences compared to previously reported values at 9.4 T were discernible. The T1 values reported here will be useful for the quantification of metabolites and signal-to-noise optimization in human brain at 7 T. Magn Reson Med 69:931-936, 2013. © 2012 Wiley Periodicals, Inc.

Prediction of functional outcome 18 months after a first psychotic episode: a proton magnetic resonance spectroscopy study.

CONTEXT: Recent magnetic resonance imaging studies have attempted to relate volumetric brain measurements in early schizophrenia to clinical and functional outcome some years later. These studies have generally been negative, perhaps because gray and white matter volumes inaccurately assess the underlying dysfunction that might be predictive of outcome. OBJECTIVE: To investigate the predictive value of frontal and temporal spectroscopy measures for outcome in patients with first-episode psychoses. DESIGN: Left prefrontal cortex and left mediotemporal lobe voxels were assessed using proton magnetic resonance spectroscopy to provide the ratio of N-acetylaspartate (NAA) and choline-containing compounds to creatine and phosphocreatine (Cr) (NAA/Cr ratio). These data were used to predict outcome at 18 months after admission, as assessed by a systematic medical record audit. SETTING: Early psychosis clinic. PARTICIPANTS: Forty-six patients with first-episode psychosis. MAIN OUTCOME MEASURES: We used regression models that included age at imaging and duration of untreated psychosis to predict outcome scores on the Global Assessment of Functioning Scale, Clinical Global Impression scales, and Social and Occupational Functional Assessment Scale, as well as the number of admissions during the treatment period. We then further considered the contributions of premorbid function and baseline level of negative symptoms. RESULTS: The only spectroscopic predictor of outcome was the NAA/Cr ratio in the prefrontal cortex. Low scores on this variable were related to poorer outcome on all measures. In addition, the frontal NAA/Cr ratio explained 17% to 30% of the variance in outcome. CONCLUSIONS: Prefrontal neuronal dysfunction is an inconsistent feature of early psychosis; rather, it is an early marker of poor prognosis across the first years of illness. The extent to which this can be used to guide treatment and whether it predicts outcome some years after first presentation are questions for further research.

A double-quadrature radiofrequency coil design for proton-decoupled carbon-13 magnetic resonance spectroscopy in humans at 7T

Purpose Carbon-13 magnetic resonance spectroscopy (13C-MRS) is challenging because of the inherent low sensitivity of 13C detection and the need for radiofrequency transmission at the 1H frequency while receiving the 13C signal, the latter requiring electrical decoupling of the 13C and 1H radiofrequency channels. In this study, we added traps to the 13C coil to construct a quadrature-13C/quadrature-1H surface coil, with sufficient isolation between channels to allow simultaneous operation at both frequencies without compromise in coil performance. Methods Isolation between channels was evaluated on the bench by measuring all coupling parameters. The quadrature mode of the quadrature-13C coil was assessed using in vitro 23Na gradient echo images. The signal-to-noise ratio (SNR) was measured on the glycogen and glucose resonances by 13C-MRS in vitro, compared with that obtained with a linear-13C/quadrature-1H coil, and validated by 13C-MRS in vivo in the human calf at 7T. Results Isolation between channels was better than â^'30 dB. The 23Na gradient echo images indicate a region where the field is strongly circularly polarized. The quadrature coil provided an SNR enhancement over a linear coil of 1.4, in vitro and in vivo. Conclusion It is feasible to construct a double-quadrature 13C-1H surface coil for proton decoupled sensitivity enhanced 13C-NMR spectroscopy in humans at 7T. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.