Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress.

Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs.

Abdominal CT during pregnancy: a phantom study on the effect of patient centring on conceptus radiation dose and image quality.

OBJECTIVES: To investigate the effect of patient centring on conceptus radiation dose and image quality in abdominal CT during pregnancy. MATERIAL AND METHODS: Three anthropomorphic phantoms that represent a pregnant woman at the three trimesters of gestation were subjected to a routine abdominal CT. Examinations were performed with fixed mAs (mAsf) and with the automatic exposure control system (AEC) activated. The percent reduction between mAsf and modulated mAs (mAsmod) was calculated. Conceptus dose (Dc) was measured using thermoluminencent dosimeters. To study the effect of misplacement of pregnant women on Dc, each phantom was positioned at various locations relative to gantry isocentre. Image quality was evaluated on the basis of image noise, signal-to-noise ratio, and contrast-to-noise ratio. RESULTS: The maximum reduction between mAsf and mAsmod was 59.8 %, while the corresponding DC reduction was 59.3 %. DC was found to decrease by up to 25 % and 7.9 % for phantom locations below and above the isocentre, respectively. Image quality deteriorated when AEC was activated, and it was progressively improved from lower to higher than the isocentre locations. CONCLUSION: Centring errors do not result in an increase in Dc. To maintain image quality, accurate centring is required. KEY POINTS: • AEC activation reduces conceptus radiation dose at all gestational stages. • Patients should be accurately aligned at the gantry isocenter. • Patient centring deserves increased attention in clinical practice. • Pregnant patient centring errors do not considerably affect conceptus dose.

The effect of surgically assisted rapid maxillary expansion on sleep architecture: an exploratory risk study in healthy young adults.

Maxillary transverse deficiencies (MTD) cause malocclusions. Rapid maxillary expansion treatment is commonly used treatment for correcting such deficiencies and has been found to be effective in improving respiration and sleep architecture in children with obstructive sleep apnoea (OSA). However, thus far, the effect of surgically assisted rapid maxillary expansion (SARME) treatment on sleep architecture and breathing of normal subjects has not been assessed. We hypothesised that sleep quality will improve after maxillary expansion treatment. The objective of this study is to access the effect of maxillary expansion treatment on sleep structure and respiratory functions in healthy young adults with severe MTD. This is a prospective and exploratory clinical study. Twenty-eight consecutive young adult patients (15 males and 13 females, mean age 20·6 ± 5·8 years) presenting with severe MTD at the orthodontic examination were recruited into the study. All the participants underwent a standardised SARME procedure (mean expansion 6·5 ± 1·8 and 8·2 ± 1·8 mm, intercanine and intermolar distance, respectively) to correct malocclusion caused by MTD. An overnight in-laboratory polysomnography, before and after the treatment, was performed. The mean follow-up time was 9 months. The main outcome parameters were the changes in sleep architecture, including sleep stages, arousals, slow-wave activity (SWA) and respiratory variables. Before surgery, young adult patients with MTD presented no evidence of sleep breathing problems. At baseline sleep recording, 7 of 28 (25%) had apnoea-hypopnoea index (AHI) ≥ 5 events per hour. No negative effect of the SARME was observed in questionnaires or sleep laboratory parameters. In the patients with a higher baseline AHI (AHI ≥ 5 h of sleep), we observed a reduction in AHI after surgical treatment (P = 0·028). SARME did not have a negative effect on any sleep or respiration parameters in healthy young individuals with MTD. It normalised the breathing index in the patients with a mild AHI index.

Technical note: a fast laser-based optical-CT scanner for three-dimensional radiation dosimetry.

PURPOSE: To introduce a novel laser-based optical-CT scanner for the readout of three-dimensional (3D) radiation dosimeters. METHODS: The scanner employs a diode laser, a cylindrical lens, a motorized linear rail, a rotation stage, and a charge-coupled device camera. The scanner operates in a translate-rotate fashion and may be set up in two configurations depending on the orientation of the cylindrical lens. The attenuation coefficient versus dose response was determined for a normoxic N-vinylpyrrolidone based polymer gel dosimeter. Cylindrical dosimeters, 2 cm diameter, were homogenously irradiated to known doses up to 60 Gy using a 6 MV linear accelerator. For a test irradiation, a 5 cm diameter dosimeter was irradiated along its cylindrical axis using a rectangular 1 cm x 1 cm irradiation beam. The dose readout of this scanner was compared to the corresponding readout of a common wide illumination and area detector optical-CT scanner. RESULTS: The attenuation coefficient versus dose response of the laser-based system was found to be linear up to 60 Gy (r2 = 0.997) compared to the wide field illumination based optical-CT scanner, which exhibits linearity up to 32 Gy (r2 = 0.996). The noise in the reconstructed attenuation coefficient maps was +/- 7.2 x 10(-2) mm(-1) versus +/- 9.5 x 10(-3) mm(-1) for the laser-based system and the wide field illumination system, respectively. CONCLUSIONS: We have developed a novel laser-based optical-CT scanner, which is capable of generating fast 3D dosimetric data using a scattering polymer gel dosimeter. Our data demonstrate that the dose readout of this scanner preserves the advantage of existing laser-based optical-CT scanners in providing measurements, which are minimally affected by scattered light. For accurate reconstruction of the attenuation coefficients, noise reduction techniques need to be applied.

Accuracy of multislice CT angiography for the assessment of in-stent restenoses in the iliac arteries at reduced dose: a phantom study.

OBJECTIVE: We investigated the potential of low-dose CT angiography for accurate assessment of in-stent restenoses (ISRs) of the iliac artery. METHOD: A Rando anthropomorphic phantom (Alderson Research Labs, Stanford, CA), custom-made wax simulating hyperplastic tissue and a nitinol stent were used to simulate a patient with clinically relevant iliac artery ISRs. The cylindrical lumen was filled with a solution of iodine contrast medium diluted in saline, representing a patient's blood during CT angiography. The phantom was subjected to standard- and low-dose angiographic exposures using a modern multidetector (MD) CT scanner. The percentage of ISR was determined using the profile along a line normal to the lumen axis on reconstructed images of 2 and 5 mm slice thickness. Percentage ISRs derived using the standard- and low-dose protocols were compared. In a preliminary study, seven patients with stents were subjected to standard- and low-dose MDCT angiography during follow-up. The resulting images were assessed and compared by two experienced radiologists. RESULTS: The accuracy in measuring the percentage ISR was found to be better than 12% for all simulated stenoses. The differences between percentage ISRs measured on images obtained at 120 kVp/160 mAs and 80 kVp/80 mAs were below 6%. Patient image sets acquired using low-exposure factors were judged to be of satisfactory diagnostic quality. The assessment of ISR did not differ significantly between image sets acquired using the standard factors and those acquired using the low-exposure factors, although the mean reduction in patient effective dose was 48%. CONCLUSION: A reduction in exposure factors during MDCT angiography of the iliac artery is possible without affecting the accuracy in the determination of ISRs.

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death.

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.

Small SRS photon field profile dosimetry performed using a PinPoint air ion chamber, a diamond detector, a novel silicon-diode array (DOSI), and polymer gel dosimetry. Analysis and intercomparison.

Small photon fields are increasingly used in modern radiotherapy and especially in IMRT and SRS/SRT treatments. The uncertainties related to small field profile measurements can introduce significant systematic errors to the overall treatment process. These measurements are challenging mainly due to the absence of charged particle equilibrium conditions, detector size and composition effects, and positioning problems. In this work four different dosimetric methods have been used to measure the profiles of three small 6 MV circular fields having diameters of 7.5, 15.0, and 30.0 mm: a small sensitive volume air ion chamber, a diamond detector, a novel silicon-diode array (DOSI), and vinyl-pyrrolidone based polymer gel dosimeter. The results of this work support the validity of previous findings, suggesting that (a) air ion chambers are not suitable for small field dosimetry since they result in penumbra broadening and require significant corrections due to severe charged particle transport alterations; (b) diamond detectors provide high resolution and rather accurate small field profile measurements, as long as positioning problems can be addressed and the necessary dose rate corrections are correctly applied; and (c) the novel silicon-diode array (DOSI) used in this study seems to be adequate for small field profile measurements overcoming positioning problems. Polymer gel data were assumed as reference data to which the other measurement data were compared both qualitatively and quantitatively using the gamma-index concept. Polymer gels are both phantom and dosimeter, hence there are no beam perturbation effects. In addition, polymer gels are tissue equivalent and can provide high-spatial density and high-spatial resolution measurements without positioning problems, which makes them useful for small field dosimetry measurements. This work emphasizes the need to perform beam profile measurements of small fields (for acceptance, commissioning, treatment planning systems data feed, and periodic quality assurance purposes) using more than one dosimetric method. The authors believe this to be a safe way towards the reduction of the overall uncertainty related to SRS/SRT treatments.

Determination of the weighted CT dose index in modern multi-detector CT scanners.

The aim of the present study was to (a) evaluate the underestimation in the value of the free-in-air (CTDI(air)) and the weighted CT dose index (CTDI(w)) determined with the standard 100 mm pencil chamber, i.e. the CTDI(100) concept, for the whole range of nominal radiation beam collimations selectable in a modern multi-slice CT scanner, (b) estimate the optimum length of the pencil-chamber and phantoms for accurate CTDI(w) measurements and (c) provide CTDI(w) values normalized to free-in-air CTDI for different tube-voltage, nominal radiation beam collimations and beam filtration values. The underestimation in the determination of CTDI(air) and CTDI(w) using the CTDI(100) concept was determined from measurements obtained with standard polymethyl-methacrylate (PMMA) phantoms and arrays of thermoluminescence dosimeters. The Monte Carlo N-Particle transport code was used to simulate standard CTDI measurements on a 16-slice CT scanner. The optimum pencil-chamber length for accurate determination of CTDI(w) was estimated as the minimum chamber length for which a further increase in length does not alter the value of the CTDI. CTDI(w)/CTDI(air) ratios were determined using Monte Carlo simulation and the optimum detector length for all selectable tube-voltage values and for three different values of beam filtration. To verify the Monte Carlo results, measured values of CTDI(w)/CTDI(air) ratios using the standard 100 mm pencil ionization chamber were compared with corresponding values calculated with Monte Carlo experiments. The underestimation in the determination of CTDI(air) using the 100 mm pencil chamber was less than 1% for all beam collimations. The underestimation in CTDI(w) was 15% and 27% for head and body phantoms, respectively. The optimum detector length for accurate CTDI(w) measurements was found to be 50 cm for the beam collimations commonly employed in modern multi-detector (MD) CT scanners. The ratio of CTDI(w)/CTDI(air) determined using the optimum detector length was found to be independent of beam collimation. Percentage differences between measured and calculated corresponding CTDI(w)/CTDI(air) ratios were always less than 8% for head and less than 5% for body PMMA phantoms. In conclusion, the CTDI(air) of MDCT scanners may be measured accurately with a 100 mm pencil chamber. However, the CTDI(100) concept was found to be inadequate for accurate CTDI(w) determination for the wide beam collimations commonly used in MDCT scanners. Accurate CTDI(w) determination presupposes the use of a pencil chamber and PMMA phantoms at least 50 cm long.

An evaluation of the dosimetric performance characteristics of N-vinylpyrrolidone-based polymer gels.

The aim of this work was to investigate the dosimetric performance properties of the N-vinylpyrrolidone argon (VIPAR) based polymer gel as a dosimetric tool in clinical radiotherapy. VIPAR gels with a larger concentration of gelatin than the standard recipe were manufactured and irradiated up to 68 Gy using a 6 and 18 MV linear accelerator. Using MRI, the R2-dose response was recorded at different imaging sessions within a 34 day time period post-irradiation. The R2-dose response was found to be linear between 5 and 68 Gy. Although dose sensitivity did not show significant variation with time, the measured R2-dose values showed an increasing trend, which was less evident beyond 17 days. At one day post-irradiation, calculated dose standard uncertainties at 20 Gy and 56 Gy were 2.2% and 1.7%, providing a dose resolution of 0.45 Gy and 0.97 Gy, respectively. Although these values fulfilled the 2% limit of ICRU, when gels were imaged at one day post-irradiation, it was shown that the temporal evolution of the R2 values deteriorated the per cent standard uncertainty and the dose resolution by approximately 57%, when imaged 17 days post-irradiation. Variation in the coagulation temperature of the gels did not impact the R2-dose sensitivity. This study has shown that the VIPAR gel has the properties of a dosimetric tool required in clinical radiotherapy, especially in applications where a wide dose dynamic range is employed. For results with the lowest per cent uncertainty and the optimum dose resolution, the dosimetry gels used in this work should be MR scanned at one day post-irradiation. Furthermore, a preliminary study on the R2-dose response of a new normoxic N-vinylpyrrolidone-based polymer gel showed that it could potentially replace the traditional VIPAR gel formulation, while preserving the wide dynamic dose response inherent to that monomer.

Experimental determination of the effect of detector size on profile measurements in narrow photon beams.

The aim of this work is to investigate experimentally the detector size effect on narrow beam profile measurements. Polymer gel and magnetic resonance imaging dosimetry was used for this purpose. Profile measurements (Pm(s)) of a 5 mm diameter 6 MV stereotactic beam were performed using polymer gels. Eight measurements of the profile of this narrow beam were performed using correspondingly eight different detector sizes. This was achieved using high spatial resolution (0.25 mm) two-dimensional measurements and eight different signal integration volumes A X A X slice thickness, simulating detectors of different size. "A" ranged from 0.25 to 7.5 mm, representing the detector size. The gel-derived profiles exhibited increased penumbra width with increasing detector size, for sizes >0.5 mm. By extrapolating the gel-derived profiles to zero detector size, the true profile (Pt) of the studied beam was derived. The same polymer gel data were also used to simulate a small-volume ion chamber profile measurement of the same beam, in terms of volume averaging. The comparison between these results and actual corresponding small-volume chamber profile measurements performed in this study, reveal that the penumbra broadening caused by both volume averaging and electron transport alterations (present in actual ion chamber profile measurements) is a lot more intense than that resulted by volume averaging effects alone (present in gel-derived profiles simulating ion chamber profile measurements). Therefore, not only the detector size, but also its composition and tissue equivalency is proved to be an important factor for correct narrow beam profile measurements. Additionally, the convolution kernels related to each detector size and to the air ion chamber were calculated using the corresponding profile measurements (Pm(s)), the gel-derived true profile (Pt), and convolution theory. The response kernels of any desired detector can be derived, allowing the elimination of the errors associated with narrow beam profile measurements.

Activated peripheral blood and endothelial cells in thalassemia patients.

BACKGROUND AND OBJECTIVES: Thalassemia patients have alterations in the expression of some activation and adhesion molecules on peripheral blood lymphocytes. We studied cell surface antigens on peripheral blood cells associated with the activation of these cells and soluble molecules produced by activated endothelium. DESIGN AND METHODS: We investigated the expression of CD11b, CD18, CD35, CD43, CD44, and CD69 on the peripheral blood monocytes, Cd11b, CD18, CD35, CD43, CD44, CD67 on peripheral blood neutrophils and CD38 and CD69 on peripheral blood lymphocytes. We studied 68 transfusion-dependent thalassemics (group A), 10 transfusion non-dependent thalassemics (group B), 18 beta-thalassemia carriers (group C), and 28 normal individuals. Relative fluorescence intensity was used to determine the antigen density. Analysis was performed with an EPICS ELITE flow cytometer. Furthermore, soluble intercelullar adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), and E-selectin, tumor necrosis factor (TNF) alpha, and interleukin (IL) 1beta were measured in the plasma of patients by enzyme-linked immunometric assay. RESULTS: The expression of CD11b, CD18, and CD69 on the monocytes of group A was significantly greater than in groups B and C and in controls, while CD44 was significantly downregulated in group A. CD11b, CD18, CD35, CD44, and CD67 on the surface of neutrophils and CD38 and CD69 on the surface of lymphocytes were also overexpressed in group A. CD44 was downregulated on the monocytes and upregulated on the neutrophils of the patients compared to controls. The levels of sICAM-1, sVCAM-1, E-selectin, TNF-alpha, and IL-1beta in the serum of patients in groups A and B were higher than those in group C and the controls. CONCLUSION: Endothelial activation markers are significantly increased in thalassemia patients, and activated blood cells circulate in the peripheral blood. These may be related to the vascular complications in these patients and might be useful markers for the follow-up of the vascular disease.

Patient dose reduction in CT examinations by optimising scanogram acquisition.

The objective was to determine the optimum settings of the scanogram performed in computed tomography (CT) examinations for scan localisation. Head, abdomen and thorax scanograms were performed on a Rando anthropomorphic phantom using various selectable combinations of tube voltage and tube current values. Thermoluminescence dosemeters were used to obtain entrance skin dose data. Effective dose was estimated using normalised organ dose data provided by the National Radiological Protection Board. One hundred and twelve head, 114 thoracic and 111 abdominal patient scanograms were obtained with lower settings than those recommended by the operator's manual. Scanogram sufficiency was assessed by four observers. Head and thoracic scanograms obtained with 80 kV/50 mA and abdominal scanograms obtained with 80 kV/75 mA were found to be acceptable, even though the operator's manual recommendation was 120 kV/100 mA. Thus, the scanogram effective dose was reduced by 72%, 84% and 88% for head, thorax and abdomen examination respectively. Effective dose from a complete CT examination may be reduced by up to 3.5% without any subsequent image quality degradation of the diagnostically important sectional images.

Broadband ultrasound attenuation imaging: influence of location of region of measurement.

The aim of the study was to investigate the effect of three different regions of interest (ROIs) varying in size and shape on broadband ultrasound attenuation (BUA) measurements of the calcaneus. Two hundred and sixty-five postmenopausal Caucasian women participated in this study. In 43 women osteoporotic fractures were documented on spinal radiographs. Bone mineral density (BMD) measurements of the lumbar spine and the femur were made using dual-energy X-ray absorptiometry. BUA measurements were obtained at a circular ROI automatically determined by the imaging system (ROIc), at a manually traced irregular ROI encompassing the posterior part of the calcaneus (ROIi), and at an anatomical square ROI located in the posterior part of the calcaneus (ROIs). Reproducibility was better in ROIc than in ROIi and ROIs. High correlations were found between BUA measurements with ROIc and ROIs (r = 0.981, P < 0.0001) as well as between those with ROIc and ROIi (r = 0.965, P < 0.0001). There were no significant differences between the correlations of BUA with axial BMD at ROIc compared with ROIi and ROIs. No significant difference was found between the areas under the ROC curve at ROIi, ROIc, and ROIs for women with fractures. The results show that superior reproducibility makes ROIc the most appropriate region of BUA measurement in a comparison with ROIi and ROIs.

Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts.

We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.

The bicipital groove as a landmark for orientation of the humeral prosthesis in cases of fracture.

We studied 45 dry cadaveric humeri to determine whether the bicipital groove of the humerus can be used as a landmark for a proper, individualized orientation of a humeral prosthesis, especially in the case of a fracture. We performed 3 computed tomography sections (at a level just below the lower portion of the head, at the middle of the humeral head, and at a distance 5 cm below the first section), and we used special software for 3-dimensional image processing. To reproduce the individual posterior version of the head, when a humeral prosthesis is implanted for fracture, the lateral fin of the prosthesis should be a mean distance 5.2 +/- 2.6 mm (-1.5 to 10.7 mm) from the posterior edge of the bicipital groove. If the lateral fin of the humeral prosthesis seats just behind the posterior edge of the bicipital groove, a difference of -6.3 degrees to 41.7 degrees from the normal posterior version occurs. A new, simple methodology for an individualized posterior version of a humeral prosthesis in cases of fracture is proposed. We applied this in 6 consecutive patients with fracture of the humeral head that required hemiarthroplasty.

The generation of active oxygen species differs in tobacco and grapevine mesophyll protoplasts.

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.

Decreased expression of c-myc oncoprotein by peripheral blood mononuclear cells in thalassaemia patients receiving desferrioxamine.

Desferrioxamine (DFX) is an iron chelation agent widely used in the treatment of transfusional iron overload in patients with thalassaemia major and other severe refractory anaemias. DFX has been shown to induce inhibition of DNA synthesis and apoptosis in vitro; however, the molecular targets of DFX action are not well known. The c-myc proto-oncogene is involved in a number of cellular processes including proliferation, differentiation and apoptotic cell death. We have examined the expression of c-myc in peripheral blood mononuclear cells from 71 patients with homozygous beta-thalassaemia in regular transfusion and iron chelation therapy with DFX, 5 non-transfusion, non-chelation-dependent thalassaemic patients, and 15 healthy volunteers using an APAAP immunocytochemical method. We have found that mononuclear cells from thalassaemic patients receiving DFX express significantly lower levels of c-myc protein compared to control healthy volunteers or thalassaemics receiving no DFX. In vitro treatment of HL60 or K562 leukaemic cells with 100 microliters DFX also induced a rapid decrease in c-myc mRNA and protein levels, followed by apoptosis and inhibition of DNA synthesis. These effects were blocked by simultaneous addition of ferric chloride. Our data suggest that deprivation of cellular iron induces downregulation of c-myc expression in vitro and in vivo and may influence haemopoietic cell growth and survival.