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We are report on a distributed online coincidence detection implementation that we have recently added to the miniPET-II small animal PET scanner. The implementation uses standard Ethernet and IP multicasting techniques, therefore no architectural changes were necessary to the existing system. For 2D reconstruction the implementation scales with the number of detectors in the system, so it can be used for larger ring configurations, too. The online coincidence detection makes the system suitable for real time imaging applications.

The PET technique is widely used in human clinical studies and recent developments of image resolution has made it suitable for small animal research. The second generation of our PET scanner consists of 12 detector modules and has a field of view large enough to image mice and rats. Parameters of the incoming data are extracted by Digital Signal Processing in the detector modules and a System-on-Module is used to transmit the data through an Ethernet network for storage and reconstruction. The experimental scanner setup described in this paper was constructed in order to investigate the applicability of the developed detector modules in a full ring small animal PET camera. The preliminary results of the system are also presented.

In vivo imaging of small laboratory animals is a valuable tool in the development of new drugs. For this purpose, miniPET, an easy to scale modular small animal PET camera has been developed at our institutes. The system has four modules, which makes it possible to rotate the whole detector system around the axis of the field of view. Data collection and image reconstruction are performed using a data acquisition (DAQ) module with Ethernet communication facility and a computer cluster of commercial PCs. Performance tests were carried out to deter-mine system parameters, such as energy resolution, sensitivity and noise equivalent count rate. A modified GEANT4-based GATE Monte Carlo software package was used to simulate PET data analogous to those of the performance measurements. GATE was run on a Linux cluster of 10 processors (64 bit, Xeon with 3.0 GHz) and controlled by a SUN grid engine. The application of this special computer cluster reduced the time necessary for the simulations by an order of magnitude. The simulated energy spectra, maximum rate of true coincidences and sensitivity of the camera were in good agreement with the measured parameters.

We present a new detector module developed for miniPET-II, the second generation of the miniPET small animal PET scanners. The improved module features new hardware components for better performance: LySO crystal material, increased number of crystal segments, Hamamatsu H9500 PSPMT, Xilinx Virtex-4 FPGA and Gigabit Ethernet. However, the principle of operation is the same: no hardware coincidence detection is implemented, data is acquired in list mode and transfered over an Ethernet network. The resulting new module is more suitable for full ring configurations.

Polar map display (PM) is a comprehensive interpretation of the left ventricle. This is a non-rigid registration of the left ventricle originally for the visual and quantitative analysis of tomographic myocardial perfusion scintigrams. In this scheme the maximal-count circumferential profiles of well-defined short- and long-axis planes are plotted to a map showing the distribution of the perfusion tracer onto a two-dimensional polar representation. The usual coronary artery distribution is often indicated on the PMs of SPECT studies by referring to the regions of the three main coronary branches, nevertheless, the individual variations may differ extensively. We set out to develop an Access (Microsoft)-based computer program that permits an integrated evaluation of the imaging results (coronary angiography, echocardiography and SPECT) on patients with coronary artery disease. This semi-quantitative registration of the coronary tree to a PM focused on the relation between the supplying coronary branches and the myocardial regions of the 16-segment left ventricular evaluating model. All the recorded anatomical and functional data were related to these 16 left ventricular segments, which allowed the direct comparison and holistic synthesis of the results. Two projections were taken into consideration for generation of the coronary PM: from the right anterior oblique projections, the left anterior descendent (LAD)/right coronary artery (RCA) border was assessed through the comparison of the left and right coronary angiograms. The terminations of the visually detected end-arteries showed the separation of the myocardial beds supplied by the two branches. The border of the myocardial beds on the polar map was determined on the "vertical axis" of the local coordinate system. The RCA/ left circumflex (LCx) separation can be determined from the left anterior oblique view. In this projection, the left ventricular septal edge was delineated by the LAD, while the LCx indicated the lateral epicardial surface. The individual coronary artery circulation was typified from among 12 variations in the Holistic Coronary Care program. With this determination of the individual coronary circulation, the lesion-associated segments are generated automatically by the software. The lesion-associated regions are defined as the myocardial bed of a diseased artery distal to the lesion. The PMs generated from the coronary angiographic results were compared with those of 99Tc-labelled MIBI single photon emission computed tomography (SPECT) in order to test the accuracy of the localizing method. The overlap between the segments associated with the coronary lesion and the stress perfusion defects (

An easy to scale up modular PET scanner was developed for imaging small animals. Energy resolution, spatial resolution and count rate performance were determined as system parameters. The configuration provided an average energy resolution of 19.6 % and the image resolution ranges was 1.5 to 2.3 mm in radial direction. The sensitivity of the miniPET was 1.08 cps/kBq as determined using a point source. In addition, results of rat brain scan performed with FDG are given to characterize imaging capability of the system. The displayed data document that the miniPET scanner supports good quality brain imaging of small animals

Aim To establish the effects of Na+/Ca2+ exchanger (NCX) blockers on 2-[18F]fluoro-2-deoxy- d -glucose (18FDG) and 11C-choline accumulation in different cancer cells. Methods The tumor cells were incubated with NCX inhibitors, and the uptakes of 18FDG and 11C-choline were measured. Flow cytometric measurements of intracellular Ca2+ and Na+ concentrations were carried out. The presence of the NCX antigen in the cancer cells was proved by Western blotting, flow cytometry and confocal laser scanning microscopy. Results The NCX is expressed at a noteworthy level in the cytosol and on the cytoplasmic membrane of the examined cells. Incubation of the cells with three chemically unrelated NCX blockers (bepridil, KB-R7943 or 3′,4′-dichlorobenzamil hydrochloride) resulted in an increase in the intracellular Ca2+ concentration, with a simultaneous decrease in the intracellular Na+ concentration. The treatment with the NCX inhibitors increased the energy consumption of the tumor cells by 50–100%. Thapsigargin abolished the NCX-induced 18FDG accumulation in the cells. The NCX blockers applied decreased the 11C-choline accumulation of all the investigated cancer cells by 60–80% relative to the control. Conclusion A possible masking effect of NCX medication must be taken into consideration during the diagnostic interpretation of PET scans.

Engineers specialized in multimodal tomography regularly face a wide scale of programming tasks requiring an integrated software system to ensure cost efficiency. Accordingly, a software development framework has been worked out comprising libraries for cluster-based data acquisition, image reconstruction, management of data files and complex multimodal volumetric visualization. This framework enabled us to develop complex software for our miniPET project [Kis et al., 2005], This software contains a graphical application integrating data acquisition, cluster monitoring, event sorting, image reconstruction, interactive image processing tools for advanced multimodal visualization. It also contains utilities to solve these tasks without graphical user interface. The components of our acquisition program can run on embedded Linux systems making new ways to develop any other types of data acquisition software that uses embedded Linux systems. A versatile development framework is developed containing specific libraries and special file formats that support multimodal tomography. This framework was successfully used to elaborate our complex miniPET software.

We report on the design of a data acquisition (DAQ) module for a small animal PET camera developed at our institutes. During the design an important guideline was to develop a system which is built up from strictly identical DAQ modules, and which has no built-in hardware limitation on the maximum number of modules. The developed DAQ module comprises of an LSO scintillator crystal block, a position sensitive PMT, analog signal conditioning circuits, a digitizer, an field programmable gate array (FPGA) for digital signal processing, and a communication module through which the collected data are sent to a cluster of computers for postprocessing and storage. Instead of implementing hardware coincidence detection between the modules, we attach a precise time stamp to each event in our design, and the coincidence is determined by the data collecting computers during postprocessing. The digital CFD algorithm implemented in the FPGA gives a time resolution of 2 ns FWHM for real detector signals

The pharmacological effects of the neuroprotective drug vinpocetine, administered intravenously in a 14-day long treatment regime, on the cerebral blood flow and cerebral glucose metabolism in chronic ischemic stroke patients (n=13) were studied with positron emission tomography in a double-blind design. The regional and global cerebral metabolic rates of glucose (CMRglc) and cerebral blood flow (CBF) as well as vital physiological parameters, clinical performance scales, and transcranial Doppler parameters were measured before and after the treatment period in patient groups treated with daily intravenous infusion with or without vinpocetine. While the global CMRglc values did not change markedly as a result of the infusion treatment with (n=6) or without (n=7) vinpocetine, the global CBF increased and regional CMRglc and CBF values showed marked changes in several brain structures in both cases, with more accentuated changes when the infusion contained vinpocetine. In the latter case the highest rCBF changes were observed in those structures in which the highest regional uptake of labelled vinpocetine was measured in other PET studies (thalamus and caudate nucleus: increases amounting to 36% and 37%, respectively). The findings indicate that a 2-week long intravenous vinpocetine treatment can contribute effectively to the redistribution of rCBF in chronic ischemic stroke patients. The effects are most pronounced in those brain regions with the highest uptake of the drug.

We report on the design of a small animal PET scanner being developed at our institutes. The existing setup is the first version of the miniPET machine consisting of four detector modules. Each detector module consists of an 8times8 LSO scintillator crystal block, a position sensitive photomultiplier, a digitizer including a digital signal processing board and an Ethernet interface board. There is no hardware coincidence detection implemented in the system and coincidence is determined based on a time stamp attached to every event by a digital CFD algorithm. The algorithm is implemented in the digital signal processing board and generates a time stamp with a coincidence resolution of less than 2 ns. The data acquisition system is based on Ethernet network and is highly scalable in size and performance

Aim: To study the accumulation and washout kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) in MDR positive and MDR negative tumour cells and how this is modified by lipophilic P-glycoprotein ligands. Methods: The tumour cells were incubated in the presence and absence of the ligands and the uptakes of 99mTc-MIBI, rhodamine 123 and 2-[18F]fluoro-2-deoxy- d -glucose (18FDG) were measured. Results: The accumulation of 99mTc-MIBI in the tumour cells followed biphasic kinetics. Verapamil and cyclosporin A increased the membrane fluidity and significantly enhanced the 99mTc-MIBI uptake of the MDR negative cells, while the rhodamine 123 uptake was not affected. Verapamil significantly increased the uptake of rhodamine 123 and 18FDG but did not modify that of 99mTc-MIBI in the MDR positive cells. Cyclosporin A significantly increased the 18FDG uptake of the MDR positive and negative tumour cells; these effects were ouabain-sensitive. Depolarization of the cytoplasmic membrane, acidification of the extracellular medium and the administration of CCCP decreased the accumulation of 99mTc-MIBI and rhodamine 123 uptake in the tumour cells. Conclusions: Lipophilic P-glycoprotein ligands modified the biphasic accumulation kinetics of the 99mTc-MIBI uptakes of MDR negative and positive tumour cells in different and complex ways and could therefore mask the P-glycoprotein pump-dependent changes in tracer accumulation.

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3-4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.

Background We hypothesized that in patients with intracerebral tumours a subcortical metabolical shift may be present when the underlying pathology can, itself, be the epileptogenic focus. We also assumed that by studying the alterations in glucose metabolism beyond the tumour's borders we could identify a modulator area. Methods Sixty-seven patients with supratentorial brain tumour associated epilepsy were investigated interictally, in normoglycaemic conditions, by using [18F]fluorodeoxyglucose positron emission tomography (FDG PET). The studies were analysed semiquantitatively by calculating standardized uptake values and asymmetry indices. Normal subjects and patients with non-epileptic brain lesions were used as controls. Results Compared to normal controls frontal and temporal tumours showed significant changes in thalamic FDG uptake, which reflected hypometabolism of the affected side. It was noted in occipito-medial cortex in temporal tumours and in lentiform nucleus in frontal tumours as well. Comparison to lesional brains only proved that there was significant hypometabolism in lentiform nucleus in temporal tumours. Conclusions The quantified values obviously reflect biological changes. The observed subcortical hypometabolism is most likely secondary to underlying pathology. Although seizures in tumorous patients do not originate from subcortical structures their influence on cortical sites of seizure initiation could be explained by defective subcortical regulation of cortical excitability.

The cerebral projection of vestibular signaling was studied by using PET with a special differential experimental protocol. Caloric vestibular stimulation (CVS)-induced regional cerebral blood flow (rCBF) changes were investigated in two populations. Butanol perfusion scans were carried out on six healthy volunteers and on six patients following the removal of tumors from the right cerebello pontine angle. The complete loss of the vestibular function postoperatively allowed a comparison of the rCBF changes in the populations with or without this input and offered a promising functional approach whereby to delineate the cortical region most responsive to pure vestibular input. The activations by left-sided and right-sided CVS were determined for both the healthy volunteers and the patient population. Statistical analysis of the data obtained following left-sided CVS did not reveal any cerebral region for which there was a significant difference in CVS-induced response by these two populations. In the case of right-sided CVS, however, the statistical comparison of the CVS-related responses demonstrated a single contralateral area characterized by a significantly different degree of response. This cortical area corresponds to part of the cortical region described recently which can be activated by both CVS and neck vibration. It appears to be anatomically identical to the aggregate of the somatosensory area SII and the retroinsular cortex described in primates, a region identified by other investigators as an analog of the parietoinsular vestibular cortex.

P-glycoprotein (Pgp) is an ABC (ATP binding cassette) transporter that is often overexpressed in tumours, contributing significantly to their multidrug resistance. In this study, we explored whether the radiotracers used in tumour diagnostics can be used for in vivo visualisation of Pgp-related multidrug resistance. We also examined the effects of different Pgp modulators on the accumulation of these radioligands in tumours with or without Pgp expression. In a SCID BC-17 mouse model, cells of the drug-sensitive KB-3-1 (MDR(-)) and the KB-V1 Pgp-expressing (MDR(+)) human epidermoid carcinoma cell lines were inoculated to yield tumours in opposite flanks. For in vivo scintigraphic (biodistribution) and positron emission tomography (PET) examinations, the mice were injected with technetium-99m hexakis-2-methoxybutylisonitrile ((99m)Tc-MIBI), carbon-11 labelled methionine and fluorine-18 fluoro-2-deoxy- d-glucose ((18)FDG). For validation, in vitro cell studies with (99m)Tc-MIBI,( 99m)Tc-tetrofosmin, [(11)C]methionine and (18)FDG were carried out using a gamma counter. The expression and function of the MDR product were proved by immunohistochemistry and spectrofluorimetry. (99m)Tc-MIBI uptake was significantly lower in KB-V1 cells as compared with KB-3-1-derived tumours in vivo (Pgp(+)/Pgp(-) =0.61+/-0.13; P0.01) and cells in vitro (Pgp(+)/Pgp(-) =0.08+/-0.01; P0.001).()Cyclosporin A reversed (99m)Tc-MIBI uptake in the Pgp+ cells, while verapamil failed to modify it. (18)FDG uptake was significantly higher in KB-V1 tumours (Pgp(+)/Pgp(-) =1.36+/-0.05; P0.01) and cells (Pgp(+)/Pgp(- )=1.52+/-0.12; P0.001). Whereas cyclosporin A eliminated the difference between FDG uptake in MDR(+) and MDR(-) cell lines, verapamil significantly increased it. When the animals were treated with verapamil, the ratio of (99m)Tc-MIBI uptake in the MDR(+) tumours to that in the MDR(-) tumours decreased to 0.38+/-0.05 ( P0.01), while the ratio of (18)FDG uptake increased to 2.1+/-0.3 ( P0.001). There were no significant differences in the [(11)C]methionine uptake in the MDR(+) and MDR(-) tumours and cell lines, nor was [(11)C]methionine accumulation modified by cyclosporin A. Parallel administration of (18)FDG and (99m)Tc-MIBI combined with verapamil treatment seems to be a good candidate as a non-invasive marker for the diagnosis of MDR-related Pgp expression in tumours.

The coronary angiography giving the morphological data of epicardiac coronary stenoses and non-invasive tests sometimes even together are insufficient for evaluating the functional consequences of the epicardiac coronary stenoses. However, catheterisation allows us to detect the blunting of coronary flow increase during maximal vasodilatation (coronary flow reserve, CFR, e.g. by Doppler flow wire) and by measuring the translesional pressure gradient by pressure wire (fractional flow reserve, FFR) providing accurate functional assessment. According to this concept the authors have developed a complex cardiac database management program (Holistic Coronary Care, HCC). The database has been built on client-server technology where the central object is the 16-segment polar map. The system contains contextual and graphical elements for a quick data entry. By the assessment of 16 left ventricular segments on a polar map display there is a possibility for direct comparison of the invasive functional and morphologic and the non-invasive functional data. Details measured by pressure or Doppler wire are also stored in the coronary evaluating unit and the coronary segments are rendered to the supplied left ventricular segments. For the correct indication of coronary interventions (from functional and financial point of view) it is important to verify unambiguously the functionally significant coronary stenoses. To the best of our knowledge, the necessity of percutan or surgical intervention can be decided the most accurately by FFR and CFR, furthermore CFR objectively shows the failure of the coronary microvasculature.

Access (Microsoft Office)-based software was developed to register the results of different cardiological investigations (echocardiography, coronarography and SPECT) in 16 left ventricular segments in a polar map display in order to compare the anatomical and functional data. Ten patients with previous myocardial infarction were selected (mean age: 48 years, EF: 36.1/spl plusmn/7.2%). They had 14 totally or subtotally occluded coronary branches. The overlap between the occlusion-associated regions derived by angiography and the corresponding defects on MIBI SPECT, and also the echocardiographic segmental wall motion scores were evaluated. The distribution of the total of 160 segments with rest perfusion defects on MIBI SPECT and the wall motion abnormalities detected by echocardiogaphy gave positive and negative predictive values of coronary occlusion of 0.94 and 0.81 against SPECT and 0.82 and 0.76 against echocardiography, respectively. Further analysis revealed that the relative MIBI activities of the individual segments correlated significantly with the wall motion scores (r=-0.87). It is concluded that the direct comparison of tomographic imaging and echocardiography with the angiogram on the polar map display in order to measure the functional consequences of coronary artery disease may help to indicate an intervention on an individual coronary lesion.

Population averaged brain activation studies require the spatial standardization of the individual perfusion PET images. This procedure is usually supported by T1-weighted MRI images. The authors developed a segmentation technique to improve the automatized transformation of the individual MRI images into Talairach space. It was for the first time in Hungary that population averages of standardized T1-weighted MRI and [15O]-butanol PET images of the brain were created after validation of the procedure using data from 23 healthy volunteers. The newly developed method offers a solution for the automatized processing of primary data from brain activation experiments prior to statistical analysis.

Ruptured aneurysms cause subarachnoidal hemorrhage (SAH) that may or may not result in neurological sequelae. Acute SAH has been shown to reduce cerebral blood flow and metabolism. An important question is whether or not the SAH induced vasospasm without clinical symptoms leads to cerebral ischemia? Using postoperative [18F]-fluorodeoxyglucose positron emission tomography in patients with vasospasm diagnosed by transcranial Doppler without clinical changes, hypermetabolism was detected as an indicator of diffuse compensated ischemia in 6 cases. In 2 patients with neurological deficit reduced glucose metabolism was observed in the frontal and temporal-basal cortex.

A 11C labeled selective adenosine A2A antagonist, (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[11C]-methylxanthine [(11C)-CSC] was prepared reacting (E)-8-(3-chlorostyryl)-1,3,-dimethylxanthine and [11C]-methyl iodide. A primary evaluation of [11C]-CSC as a potential tracer for mapping adenosine A2A receptors by positron emission tomography (PET) was also presented. Autoradiographic studies were carried out on Swiss mice. A high level accumulation of radioactivity was observed in the striatum and medulla oblongata in accordance with previous findings on the specific spatial distribution of A2A adenosine receptors. Dynamic PET studies on rabbits showed a fast brain uptake of CSC, reaching a maximum in less than 2 minutes. Competition experiments with the unlabeled ligand proved [11C]-CSC to bind specifically to the appropriate receptor.

Results of 157 PET (positron emission tomography) studies in 102 patients with known or presumed intracranial (ic.) tumors are reviewed. In 106 studies [18F]-2-fluoro-2-deoxy-D-glucose (FDG) and in 51 cases [11C]-methionine (MET) was used as the PET tracer. CT and MRI imaging accompanied the PET studies in each case. Results of pre- and/or postoperative studies are compared. PET was concluded to have a fundamental role in the precise differential diagnosis in case of ic. space-occupying pathologies since it is capable of providing information that is not obtainable or may be equivocal based solely on CT and/or MRI investigations. However, it is also mandatory to be aware of the limits of PET.

The comparative analysis of two kinetic methods most frequently applied for the quantization of the results of FDG-PET brain scans was performed applying PET investigations and computer simulations. The analysis of the measured data and computer simulation demonstrated that the applicability of the results of SUV-methods to estimate glucose metabolic rates (GMR) as calculated by the Phelps-method (the most advanced method ) depended on the selected cerebral region. The distorted estimation was explained by the fact that the simpler models neglect dephosphorylation of the FDG-6P, and they also disregard the contribution of the intravascular activity to the tissue radioactivity. The simulations revealed the lack of a general and close positive correlation between SUV and GMR data; under special circumstances they might even change in an inverse manner.

The PET technique has been available for myocardial viability studies in Hungary since 1994. The early comparative results showed that FDG-PET identified more potentially reversible myocardial damages than [201Tl]-scintigraphy, defining the viability either by preserved metabolic activity of 50% or by metabolism-perfusion mismatch. According to the follow up of revascularized patients in our cases, FDG-PET had a positive predictive value for functional recovery at 80%. In our opinion, it is the detection of glucose metabolism that predicts with the greatest accuracy segments of hibernating myocardial tissue that will recover after revascularization. Unlike dobutamine echocardiography, PET can detect viable ventricular segments in the myocardium of lost contractile material often seen in the histological studies of chronically ischemic myocardium. The FDG-PET method can be regarded as the gold standard of myocardial viability.

The well known and widely used P1 adenosine agonist, 5'-N-ethyl-carboxamidoadenosine (NECA), was labelled with 18F isotope for the in vivo PET investigation of A1, A2 and A3 adenosine receptor expression. The precursor 2-[18F]fluoroethylamine was reacted with 2',3'-O-isopropylideneadenosine-5'-uronic acid. Specific activity of the [18F]-FNECA was (2.3 +/- 1.1) TBq/mmol (60 Ci/mmol). Dynamic PET measurements were carried out in rabbits to study the in vivo kinetics of the receptor saturation with the labelled ligand. The time dependent accumulation was followed up in the heart, lungs, liver, brain and testis. The radiotracer uptake was rapid and reached its maximum in less than two minutes in the heart and testes after v. injection of the radiopharmaceutical, while it took about 6 minutes in the brain, lungs and liver. High [18F]-FNECA accumulation was detected in the intestines, too. The specific binding of the [18F]-FNECA was tested in competition experiments in brain and heart sections using autoradiographic technique. The outlined synthesis provided sufficient amounts of [18F]-FNECA to map adenosine receptor expression under physiological conditions.

Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca 2+ -free hypoosmotic solutions, and significant increase in the intracellular Ca 2+ level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10 −7 to 10 −5 M Ca 2+ . Ca 2+ channel blockers such as verapamil and ω-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca 2+ influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca 2+ channels, leading to Ca 2+ influx at the initiation of carp sperm motility.

A 11C labeled selective adenosine A2A antagonist, (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[11C]methylxanthine ([11C]CSC) was prepared by the reaction of (E)-8-(3-chlorostyryl)-1,3,-dimethylxanthine and [11C]methyl iodide. The decay-corrected radiochemical yield was 32.3% with a radiochemical purity of 99%, a specific activity of 1.85–5.55 GBq/μmol and a preparation time of 1 h. A primary evaluation of [11C]CSC as a potential tracer for mapping adenosine A2A receptors by positron emission tomography (PET) is also presented. Biodistribution and autoradiographic studies were carried out on Swiss mice and domestic rabbits. In mice the lung showed the highest uptake at 10 min after i.v. injection, followed by the liver, kidney, heart and brain. Inside the brain a high level of radioactivity accumulated in the striatum, in accordance with previous findings on the specific spatial distribution of A2A adenosine receptors and also in the medulla oblongata. Dynamic PET studies on rabbits showed a fast brain uptake of CSC, reaching a maximum in less then 2 min. On the basis of competition experiments with the unlabeled ligand [11C]CSC proves to bind specifically to the appropriate receptor.

In vivo glucose metabolism of the brain was examined using the [18F] fluoro-deoxy-D- glucose (FDG) analogue of the glucose. A relatively high number (20–25) of volumes of interest (VOI) were defined on the PET images. Data of the kinetic studies belonging to these VOI-s were evaluated using the standard uptake value (SUV) method, or calculating glucose metabolic rate (GMR) applying Patlak analysis (PA); and three-compartment model with nonlinear regression (TCM). Correlation of data for all pairs of calculated parameters showed that accurate determination of glucose utilisation in anatomic regions of white matter and those located next to large blood vessels require application of the three-compartment tracer kinetic model calculations with no simplifying assumptions.

The effects of vinpocetine (Cavinton) on the cerebral glucose metabolism of chronic stroke patients are studied with positron emission tomography. The regional and global cerebral metabolic rates of glucose (CMRglu) and the kinetic constants related to them are quantified before and after single-dose intravenous vinpocetine treatment. These measurements are completed with transcranial Doppler sonography and single photon emission computed tomography to explore the possible mechanisms underlying the resulting changes in glucose uptake and metabolism in the brain. The authors' findings indicate that a single-dose vinpocetine treatment, although it does not affect significantly the regional or global metabolic rates of glucose, improves significantly the transport of glucose (both uptake and release) through the blood-brain barrier in the whole brain, the entire contralateral hemisphere, and in the brain tissue around the infarct area of the symptomatic hemisphere. These changes are in accord with increased blood flow in the entire contralateral hemisphere as well as decreased blood flow velocity and increased peripheral vessel resistance in the entire symptomatic hemisphere.

It is well-known that the results of PET investigations can be quantified, i.e. biochemical parameters rendered to a whole image or to a given region of interest can be determined. For this purpose, defined mathematical transformations have to be performed on the reconstructed images. From stored primary data of PET scans images can be reconstructed in terms of radioactivity concentration. True activity concentration values can also be derived from the data, as all the necessary corrections can be carried out with high precision. (distortions are produced, among others, by tissue attenuation, scattered photons, and random coincidence). Modeling the kinetics of the biochemical processes of a supposed mechanism, allows us to calculate the interrelationship between measured activity concentration and the biochemical parameters of the tracer used based on which numerical values of these parameters can be evaluated.

Recently developed flow cytometric methods (Tron et al.: Mol Immunol 27:1307-1311, 1990) to measure the intracellular pH (pHi) and intracellular potas- sium concentration in mammalian cells by using the fluorescent pH-indicator dye 28,7bis-(2-carboxy- ethyl)-5(and-6)-carboxyfluorescein (BCECF) were adopted for measuring these parameters in carp sperm. The intracellular potassium concentration of the carp sperm was 62.4 6 5.3 mM. This is very similar to the potassium concentration of the semi- nal plasma (87 6 16 mM), and it suggests a depolar- ized state of the sperm cell in the semen. An average pHi value of 7.06 6 0.11 was obtained by measuring sperm samples taken from ten animals. Changes in the ionic composition of the environment did not alter pHi. Sperm motility was initiated by transfer- ring the cells to an environment of 110 mOsm osmolality. This hypoosmotic shock induced fast changes in the membrane structure that could be reversed by restoring physiologic osmolality. Activa- tion was accompanied by a fast alkalinization of the sperm cells. This pH change was amiloride sensitive, suggesting the involvement of the Na 1 /H 1 ex- changer in the activation process. Alkalinization of acid-loaded sperm cells depended on the osmolality of the environment. Equilibrium pHiof these cells in hyperosmotic buffers was substantially lower rela- tive to cells in an isoosmotic environment. Effects of the extracellular and intracellular pH on carp sperm motility were also examined. Extracellular pH below 5.5 abolished sperm motility completely. Alkaline extracellular pH did not alter the duration of sperm motility even at extreme values (pHe 5 9.6). Dura- tion of the flagellar motion did not depend on the pHi between values of 6.5 and 8.5; however, it was significantly reduced both below and above this range. No motility was observed below pHi 5 6.0 or above pHi 5 9.5 with a 10 min incubation time at these pH values prior to activation. Effects of the extracellular and intracellular pH on sperm motility werepartiallyreversible. Cytometry27:374-382,1997.

A flow cytometric assay has been developed for determination of intracellular free potassium concentration ([K+]i). Investigated cells, loaded with the fluorescent pH indicator 2',7bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), are incubated in the presence of nigericin, and the intracellular pH is measured. The ionophore maintains the same ratio value [H+]/[K+] on both sides of the cytoplasmic membrane, so [K+]i can be evaluated from the measured intracellular pH (pHi) and the known parameters of the buffer. Application of the method revealed that the intracellular potassium concentration is significantly higher in lymphocytes than in proliferating cells of HUT-78, U266, and JY cell lines. A surprisingly low (60 mM) [K+]i concentration was observed with sperm cells of common carp. This method allows measurements on individual cells, provides data of excellent statistics, and still does not require large amounts of material. These features offer remarkable advantages over other techniques used for intracellular K+ measurements, such as steady-state fluorescence, atom absorption photometry, or energy-dispersive x-ray analysis.

The intracellular pH (pH(i)) of cells exhibiting multidrug resistance (MDR) related to the expression of the P-glycoprotein (Pgp) is often more alkaline than that of the parental cells, as also observed for the KB-V1/KB-3-1 system in this paper. The possible role of an elevated pH(i) in Pgp-related MDR has been investigated by shifting back the pH(i) of the MDR+ cells to a more acidic value using the mobile proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP). The influence of CCCP-evoked delta pH(i) on relative daunorubicin (DNR) accumulation was similar in the case of several Pgp positive and negative cell lines, in view of flow cytometric and radioactive drug accumulation studies and measuring DNR levels in the medium in a flow-through system. Our data argue against a significant effect of pH(i) on Pgp pumping efficiency. However, an indirect connection between pH(i) regulation and the MDR phenotype is suggested by the fact that acidification of the external medium in the presence of verpamil could be observed exclusively in MDR+ cells.

Membrane potential measurements using fluorescent membrane potential indicator dyes report on relative changes but usually do not result in an absolute value of the measured parameter. The method developed in this paper is based on the assumption that the negatively charged bis-oxonol distributes across the cytoplasmic membrane according to the Nernst equation. It is further supposed that the fluorescence intensity measured from a given stained cell is a single-value function of the intracellular dye concentration. The protocol suggested incorporates the construction of a calibration curve (fluorescence intensity measured from stained cells vs. extracellular dye concentration). This allows the evaluation of the membrane potential in millivolts using fluorescence readings of the cells both in the depolarized state and in the state of interest. Good agreement was found between absolute membrane potential data of human peripheral blood lymphocytes by our method and results of parallel patch clamp measurements.

A new flow cytometric method was developed for measuring the intracellular pH (pH i ) of mammalian cells using a fluorescent pH indicator dye 2′,7-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). Emission intensities (or their ratios) measured from BCECF-loaded cells can be converted into absolute pH i values using appropriate calibration curves. By comparison of several possible measuring and data evaluation procedures a double-ratio method was suggested as the most advantageous protocol to yield reliable intracellular pH data. This method allows pH i to be determined on a cell-by-cell basis corrected for cell volume and change in geometry of input—output optics of the flow cytometer. Our method applies a standard calibration curve and does not necessitate its reconstruction for each new set of measurements. Cells of the OKT-4 and OKT-8 hybridoma lines were exposed to neutron irradiation of different doses. Irradiated cells underwent a biphasic alkalinization; an instantaneous effect detected within 1.5 h was found to be intensified over 24 h. For the interpretation of data we suggest that the increase in cytoplasmic pH following neutron treatment is evoked by two mechanisms.

We have studied a bretylium tosylate induced increase of the membrane potentials of partially depolarized rat, mouse and human lymphocytes, using the potential sensitive dye, bis [1,3, dibutylbarbituric acid-(5) trimethine oxonol]. The extent of this repolarization is dose-dependent and decreased in magnitude as the temp was reduced from 37 degrees C to room temp. The repolarizing effect is inhibited by K(+)-Na(+)-pump blockers or lack of extracellular Na+. Sodium ion channel blockers are effective in abolishing repolarization only if applied prior to, or simultaneously with, bretylium. Activation of Na+/H+ exchange is not involved in the mechanism of the phenomenon as the latter is completely eliminated in the presence of 10 microM amiloride (concn of the diuretics having no measurable inhibition on the action of the exchanger). These data suggest that bretylium opens ligand- and voltage-gated Na+ channels, and repolarization occurs due to higher activity of the K(+)-Na(+)-pump stimulated by the enhanced intracellular Na+ accumulation.

Bretylium tosylate - a sodium channel opener - resulted in an increase of membrane potential of depolarized human, rat and mouse T and B lymphocytes. Flow cytometric membrane potential measurements with bis-oxonol revealed that the above hyperpolarizing effect was amiloride, ouabain, tetrodotoxin, azide and temperature sensitive. The effect showed an absolute dependence on the extracellular sodium but it was insensitive to the extracellular Ca2+ level. The voltage gating of the effect can be eliminated by either an increase of the extracellular potassium concentration or low doses of veratrin. The existence of a voltage and ligand gated sodium channel is suggested in the plasma membrane of all kinds of lymphocytes. The hyperpolarization is explained by an increased activity of the electrogenic sodium-potassium ATP-ase. Induced opening of such sodium channels may regulate the electrogenic pump activity and indirectly cell activation.

the initial results of human clinical studies demonstrated the safety and potential benefit of autologous bone marrow cells in improving left ventricular function after acute myocardial infarction (AMI). However, a fundamental problem in developing stem cell (SC)–based therapies has been the