Natural antioxidants: therapeutic prospects for cancer and neurological diseases.

An all out war is continuously occurring between oxidants and antioxidants inside the cells. This mini-review will provide an updated revision of the function of some natural compounds having main roles in antioxidant function. We will point on some phytochemicals working at two outstanding targets, tumour cells and neurons.

Eye movements of African chameleons: spontaneous saccade timing.

Despite asynchrony, saccades of left and right eyes of African chameleons had similar timing statistics. Prominent qualitative aspects of these statistics did not change if one or both eyes were masked. Evidently, an internal stochastic process regulated chameleon saccade generation.

Expanding the Capability of Microwave Multiplexed Readout for Fast Signals in Microcalorimeters.

Microwave SQUID multiplexing has become a key technology for reading out large arrays of X-ray and gamma-ray microcalorimeters with mux factors of 100 or more. The desire for fast X-ray pulses that accommodate photon counting rates of hundreds or thousands of counts per second per sensor drives system design toward high sensor current slew rate. Typically, readout of high current slew rate events is accomplished by increasing the sampling rate, such that rates of order 1MHz may be necessary for some experiments. In our microwave multiplexed readout scheme, the effective sampling rate is set by the frequency of the flux-ramp modulation (f r) used to linearize the SQUID response. The maximum current slew rate between samples is then nominally Φ 0 f r/2M in (where M in is the input coupling) because it is generally not possible to distinguish phase shifts of > π from negative phase shifts of < -π. However, during a pulse, we know which direction the current ought to be slewing, and this makes it possible to reconstruct a pulse where the magnitude of the phase shift between samples is > π. We describe a practical algorithm to identify and reconstruct pulses that exceed this nominal slew rate limit on the rising edge. Using pulses produced by X-ray transition-edge sensors, we find that the pulse reconstruction has a negligible impact on energy resolution compared to arrival time effects induced by under-sampling the rising edge. This technique can increase the effective slew rate limit by more than a factor of two, thereby either reducing the resonator bandwidth required or extending the energy range of measurable photons. The extra margin could also be used to improve crosstalk or to decrease readout noise.

A 300-mK Test Bed for Rapid Characterization of Microwave SQUID Multiplexing Circuits.

Microwave SQUID multiplexing is a promising technique for multiplexing large arrays of transition edge sensors. A major bottleneck in the development and distribution of microwave SQUID multiplexer chips occurs in the time-intensive design testing and quality assurance stages. To obtain useful RF measurements, these devices must be cooled to temperatures below 500 mK. The need for a more efficient system to screen microwave multiplexer chips has grown as the number of chips requested by collaborators per year reaches into the hundreds. We have therefore assembled a test bed for microwave SQUID circuits, which decreases screening time for four 32-channel chips from 24 h in an adiabatic demagnetization refrigerator to approximately 5 h in a helium dip probe containing a closed cycle 3He sorption refrigerator. We discuss defining characteristics of these microwave circuits and the challenges of establishing an efficient testing setup for them.

Microwave SQUID Multiplexer Demonstration for Cosmic Microwave Background Imagers.

Key performance characteristics are demonstrated for the microwave SQUID multiplexer (µmux) coupled to transition edge sensor (TES) bolometers that have been optimized for cosmic microwave background (CMB) observations. In a 64-channel demonstration, we show that the µmux produces a white, input referred current noise level of [Formula: see text] at -77 dB microwave probe tone power, which is well below expected fundamental detector and photon noise sources for a ground-based CMB-optimized bolometer. Operated with negligible photon loading, we measure [Formula: see text] in the TES-coupled channels biased at 65% of the sensor normal resistance. This noise level is consistent with that predicted from bolometer thermal fluctuation (i.e. phonon) noise. Furthermore, the power spectral density is white over a range of frequencies down to ~ 100 mHz, which enables CMB mapping on large angular scales that constrain the physics of inflation. Additionally, we report cross-talk measurements that indicate a level below 0.3%, which is less than the level of cross-talk from multiplexed readout systems in deployed CMB imagers. These measurements demonstrate the µmux as a viable readout technique for future CMB imaging instruments.

Developments in Time-Division Multiplexing of X-ray Transition-Edge Sensors.

Time-division multiplexing (TDM) is a mature scheme for the readout of arrays of transition-edge sensors (TESs). TDM is based on superconducting-quantum-interference-device (SQUID) current amplifiers. Multiple spectrometers based on gamma-ray and X-ray microcalorimeters have been operated with TDM readout, each at the scale of 200 sensors per spectrometer, as have several astronomical cameras with thousands of sub-mm or microwave bolometers. Here we present the details of two different versions of our TDM system designed to read out X-ray TESs. The first has been field-deployed in two 160-sensor (8 columns × 20 rows) spectrometers and four 240-sensor (8 columns × 30 rows) spectrometers. It has a three-SQUID-stage architecture, switches rows every 320 ns, and has total readout noise of 0.41 µΦ0/√Hz. The second, which is presently under development, has a two-SQUID-stage architecture, switches rows every 160 ns, and has total readout noise of 0.19 µΦ0/√Hz. Both quoted noise values are non-multiplexed and referred to the first-stage SQUID. In a demonstration of this new architecture, a multiplexed 1-column × 32-row array of NIST TESs achieved average energy resolution of 2.55±0.01 eV at 6 keV.

Structure/function relationship studies on the T/S residues 173-177 of rat ODC.

A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.

HOLMES: The electron capture decay of [Formula: see text]Ho to measure the electron neutrino mass with sub-eV sensitivity.

The European Research Council has recently funded HOLMES, a new experiment to directly measure the neutrino mass. HOLMES will perform a calorimetric measurement of the energy released in the decay of [Formula: see text]Ho. The calorimetric measurement eliminates systematic uncertainties arising from the use of external beta sources, as in experiments with beta spectrometers. This measurement was proposed in 1982 by A. De Rujula and M. Lusignoli, but only recently the detector technological progress allowed to design a sensitive experiment. HOLMES will deploy a large array of low temperature microcalorimeters with implanted [Formula: see text]Ho nuclei. The resulting mass sensitivity will be as low as 0.4 eV. HOLMES will be an important step forward in the direct neutrino mass measurement with a calorimetric approach as an alternative to spectrometry. It will also establish the potential of this approach to extend the sensitivity down to 0.1 eV. We outline here the project with its technical challenges and perspectives.

Role of reactive oxygen species in apoptosis: implications for cancer therapy.

Reactive oxygen species are widely generated in biological systems. Consequently humans have evolved antioxidant defence systems that limit their production. Intracellular production of active oxygen species such as *OH, O2- and H2O2 is associated with the arrest of cell proliferation. Similarly, generation of oxidative stress in response to various external stimuli has been implicated in the activation of transcription factors and to the triggering of apoptosis. Here we review how free radicals induce DNA sequence changes in the form of mutations. deletions, gene amplification and rearrangements. These alterations may result in the initiation of apoptosis signalling leading to cell death, or to the activation of several proto-oncogenes and or the inactivation of some tumour suppressor genes. The regulation of gene expression by means of oxidants, antioxidants and the redox state remains as a promising therapeutic approach. Several anticarcinogenic agents have been shown to inhibit reactive oxygen species production and oxidative DNA damage, inhibiting tumour promotion. In addition, recombinant vectors expressing radical-scavenging enzymes reduce apoptosis. In conclusion, oxidative stress has been implicated in both apoptosis and the pathogenesis of cancer providing contrived support for two notions: free radical reactions may be increased in malignant cells and oxidant scavenging systems may be useful in cancer therapy.

Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.

Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells.

Ornithine induced more than 36-fold the ornithine decarboxylase activity in confined Ehrlich ascites tumour cells after 3.5 h of continuous perifusion with 0.5 mM ornithine; arginine and glutamine also induced the activity 3- and 4-fold, respectively. The addition of cycloheximide or actinomycin D antibiotics to the perifusion medium confirmed that the regulation of the enzyme synthesis takes place at the level of translation. Perifusion in the presence of 0.5 mM ornithine and 55, 25, and 10 microM histamine suppressed the induction by 91, 53, and 35% respectively. Similar results were obtained in the presence of serotonin. Histidine also showed inhibitory effect but 5 mM histidine was required to produce 21% inhibition; other basic amino acids were ineffective.

Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing in vivo.

The antihistaminic (+/-)-chlorpheniramine significantly reduced the progression of Ehrlich carcinoma when it was administered at 0.5 mg/mouse/day from the third day on, after tumour inoculation. The ODC activity of tumour cells was diminished by 70% on day 7 after tumour transplantation, when maximum ODC activity is detected in non-treated tumour growing 'in vivo'. Northern blot analyses indicated that the inhibitory effect of this 1,4-diamine takes place at a post-transcriptional level. Results obtained from serum-free cultured cells indicated that chlorpheniramine inhibits the ODC synthesis rate.

Antioxidant enzymes and their implications in pathophysiologic processes.

Aerobic organisms possess antioxidant defense systems that deal with reactive oxygen species (ROS) produced as a consequence of aerobic respiration. Reactive oxygen is related to both, the arrest of growth and the start of cell differentiation. Low concentrations of reactive oxygen intermediates may be beneficial or even indispensable in processes such as intracellular messaging and defense against micro-organisms, but higher amounts of active oxygen may be harmful to cells and organisms. A wide array of non-enzymatic and enzymatic antioxidant defenses exists, including superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT). We describe their main characteristics and how these antioxidant enzymes work together against active oxygen. Small deviations from their physiological values may have a dramatic effect on the resistance of cells to oxidative damage to lipids, proteins and DNA. Consequently, toxic oxygen play a role in aging process as well as in a number of human diseases that we list in this review.

The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells.

The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in 'de novo' synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of 'de novo' protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC activity observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells.

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 microM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 microM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 microM N-acetyl putrescine, N-acetyl serotonin or N-omega-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistamic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55 microM 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N-N distance (6A) and the charge of one amino group are important chemical characteristics for regulatory effects.

Antioxidant enzymes and human diseases.

OBJECTIVES: To describe the importance of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase working together in human cells against toxic reactive oxygen species, their relationship with several pathophysiologic processes and their possible therapeutic implications. CONCLUSIONS: Reactive oxygen species (ROS) are involved in the cell growth, differentiation, progression, and death. Low concentrations of ROS may be beneficial or even indispensable in processes such as intracellular signaling and defense against micro-organisms. Nevertheless, higher amounts of ROS play a role in the aging process as well as in a number of human disease states, including cancer, ischemia, and failures in immunity and endocrine functions. As a safeguard against the accumulation of ROS, several nonenzymatic and enzymatic antioxidant activities exist. Therefore, when oxidative stress arises as a consequence of a pathologic event, a defense system promotes the regulation and expression of these enzymes.

Chemical and biological activity of free radical 'scavengers' in allergic diseases.

Reactive oxygen species (ROS) are generated constantly in vivo. They can lead to lipid peroxidation and oxidation of some enzymes, as well as protein oxidation and degradation. Cells possess several biological systems, defined as 'scavengers', to protect themselves from the radical-mediated damage. Immune cells may discharge their arsenal of toxic agents against host tissues, resulting in oxidative damage and inflammation. Therefore, free radical production and disturbance in redox status can modulate the expression of a variety of immune and inflammatory molecules, leading to inflammatory processes, both exacerbating inflammation and effecting tissue damage. Recently, abnormal immunity has been related to oxidative imbalance, and antioxidant functions are linked to anti-inflammatory and/or immunosuppressive properties. Currently, allergy is one of the most important human diseases. We studied the role of the primary antioxidant defence system, constituted by the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, protecting cells from toxic oxygen. We analyzed how they are involved in blood cells detoxification, and how the imbalance of reactive oxygen species is related to inflammation in allergic diseases by affecting immune cells. Finally, we discuss the published data that relates anti-free radical therapy to the management of human allergic diseases.

Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology.

Reactive Oxygen Species (ROS) are produced during normal cellular function. ROS include hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide. They are very transient species due to their high chemical reactivity that leads to lipid peroxidation and oxidation of DNA and proteins. Under normal conditions, antioxidant systems of the cell minimize the perturbations caused by ROS. When ROS generation is increased to an extent that overcomes the cellular antioxidants, the result is oxidative stress. It is now clear that several biological molecules, which are involved in cell signaling and gene regulation systems are very sensitive to redox statue of the cell. Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. The various antioxidants exert their effect by scavenging superoxide, or by activating of a battery of detoxifying/defensive proteins. The prevention of oxidation is an essential process in all the aerobic organisms, as decreased antioxidant protection may lead to cytotoxicity, mutagenicity and/or carcinogenicity. This article also focuses on the mechanisms by which antioxidants and xenobiotics induce the gene expression of detoxifying enzymes. On the other hand, small molecules that mimic antioxidant enzymes are becoming new tools for the treatment of many diseases.

Altered ornithine metabolism in tumor-bearing mice.

A flux of ornithine from the host tissues to the tumor was deduced from the concentrations of ornithine in plasma, ascitic liquid, liver and tumor cells during tumor growth. The activities of arginase and ornithine decarboxylase in both liver and tumor cells confirmed this proposed ornithine supply. Moreover, "in vitro" incubations of tumor cells showed that glutamine could be an additional source of ornithine for tumors. Finally, shortly before death, when tumor cell proliferation had ceased, altered hepatic ornithine metabolism was also detected.

Two-dimensional manual tracking of periodic movements: event and time interval analyses.

Human visuo-motor functions were analysed by means of two-dimensional manual tracking tasks. A computer-based system for visual target generation, its movement control and real-time evaluation of the subjects' performance was developed. Various types of two-dimensional periodic target movements (over circular and square trajectories with different velocities) and various settings of tracking spot-joystick relation (normal and reversed direction) were used. Tracking success was expressed in terms of: (a) incidence of tracking errors; and (b) time spent within target. Results from the experiments performed with healthy non-trained subjects have demonstrated that tracking success is lower for a square target trajectory than for the circular one, and for higher target velocity than for the lower one. Reversed two-dimensional tracking was found to be very difficult. The results based upon the two criteria of tracking success (a,b) were different for the majority of tracking variants adopted. Quantitative and qualitative differences among subjects in tracking success were found. Two-dimensional target movements were substantially more difficult than one-dimensional ones with comparable parameters (analyzed in a previous paper).