The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C.

The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.

Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C.

A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1). The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling. The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock. It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection.

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the "oxidative burst," they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.

Serum retinol and HIV-1 RNA viral load in rapid and slow progressors.

Few studies have addressed the relation between serum vitamin A levels and HIV disease progression. Thirty HIV-infected women in Rwanda were studied over a time span of 26 to 99 months. Fourteen subjects seroconverted and died of HIV-related disease at a mean of 44 months (range, 26-69 months) after their first HIV-positive test and were termed "rapid progressors," (RPs). A comparison group of 16 "slow progressors" (SPs) were HIV-positive at the time of their first HIV serology and had asymptomatic HIV infections at a mean of 96 months (range, 93-99 months) after their first HIV serology. Baseline mean serum retinol values were the same in RPs and SPs: 0.65 + 0.08 micromol/L versus 0.67 + 0.09 micromol/L (p = .7). Lower serum retinol levels were observed in RPs compared with SPs for the second and third measurements, obtained at a median of 12 and 24 months past baseline: 0.51 + 0.07 micromol/L versus 0.76 + 0.14 micromol/L (p = .3) and 0.44 + 0.09 micromol/L versus 0.64 + 0.08 micromol/L (p = .08), respectively. Median retinol levels for the third sample measurement were similar in RPs with lower viral load (LVL) and SPs (0.49 micromol/L and 0.52 micromol/L, respectively) compared with only 0.19 micromol/L in RPs with higher viral load (HVL; p = .02). A trend toward decreasing serum retinol levels and increasing HIV-1 RNA viral load was observed at the third sample measurement (p = .04). Subjects with LVL, higher serum retinol levels (> or =0.70 micromol/L), or both had more favorable rates of survival than subjects with HVL, low serum retinol levels (<0.70 micromol/L), or both. Although sample size does not permit definitive conclusions, this study demonstrates an association of high HIV load, rapid progression, and low serum retinol late but not early in disease progression.

Serum retinol and HIV-1 RNA viral load in rapid and slow progressors.

Few studies have addressed the relation between serum vitamin A level and HIV disease progression. Thirty HIV-infected women in Rwanda were studied over a time span of 26 to 99 months. Fourteen subjects seroconverted and died of HIV-related disease at a mean of 44 months (range, 26-69 months) after their first HIV-positive test and were termed "rapid progressors," (RPs). A comparison group of 16 "slow progressors" (SPs) were HIV-positive at the time of their first HIV serology and had asymptomatic HIV infections at a mean of 96 months (range, 93-99 months) after their first HIV serology. Baseline mean serum retinol values were the same in RPs and SPs: 0.65 + 0.08 mmol/L versus 0.67 + 0.09 micromol/L (p = .7). Lower serum retinol levels were observed in RPs compared with SPs for the second and third measurements, obtained at a median of 12 and 24 months past baseline: 0.51 + 0.07 mmol/L versus 0.76 + 0.14 mmol/L (p = .3) and 0.44 + 0.09 mmol/L versus 0.64 + 0.08 mmol/L (p = .08), respectively. Median retinol levels for the third sample measurement were similar in RPs with lower viral load (LVL) and SPs (0.49 mmol/L and 0.52 mmol/L, respectively) compared with only 0.19 mmol/L in RPs with higher viral load (HVL; p = .02). A trend toward decreasing serum retinol levels and increasing HIV-1 RNA viral load was observed at the third sample measurement (p = .04). Subjects with LVL, higher serum retinol levels (> or =0.70 mmol/L), or both had more favorable rates of survival than subjects with HVL, low serum retinol levels (<0.70 mmol/L), or both. Although sample size does not permit definitive conclusions, this study demonstrates an association of high HIV load, rapid progression, and low serum retinol late but not early in disease progression.

Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco.

Transgenic tobacco deficient in the H2O2-removing enzyme catalase (Cat1AS) was used as an inducible and noninvasive system to study the role of H2O2 as an activator of pathogenesis-related (PR) proteins in plants. Excess H2O2 in Cat1AS plants was generated by simply increasing light intensities. Sustained exposure of Cat1AS plants to excess H2O2 provoked tissue damage, stimulated salicylic acid and ethylene production, and induced the expression of acidic and basic PR proteins with a timing and magnitude similar to the hypersensitive response against pathogens. Salicylic acid production was biphasic, and the first peak of salicylic acid as well as the peak of ethylene occurred within the first hours of high light, which is long before the development of tissue necrosis. Under these conditions, accumulation of acidic PR proteins was also seen in upper leaves that were not exposed to high light, indicating systemic induction of expression. Short exposure of Cat1AS plants to excess H2O2 did not cause damage, induced local expression of acidic and basic PR proteins, and enhanced pathogen tolerance. However, the timing and magnitude of PR protein induction was in this case more similar to that in upper uninfected leaves than to that in hypersensitive-response leaves of pathogen-infected plants. Together, these data demonstrate that sublethal levels of H2O2 activate expression of acidic and basic PR proteins and lead to enhanced pathogen tolerance. However, rapid and strong activation of PR protein expression, as seen during the hypersensitive response, occurs only when excess H2O2 is accompanied by leaf necrosis.

Ozone-induced oxidative burst in the ozone biomonitor plant, tobacco Bel W3.

Localized cell death is a common feature of ozone phytotoxicity and is generally thought to be initiated by the strong oxidant ozone itself as well as by ozone-derived reactive oxygen intermediates (ROIs). Here we report that ozone (150 nl l(-1), 5 h) elicits cellular ROI production in the ozone-sensitive tobacco cv. Bel W3, but not in the tolerant cv. Bel B. Both cultivars exhibited a transient first maximum of apoplastic ROI accumulation followed by a comparable induction of glutathione peroxidase transcript levels. During postcultivation in pollutant-free air, a second and sustained peak of apoplastic ROI accumulation was detected only in cv. Bel W3. Histochemical staining revealed a spot-like accumulation of H(2)O(2) and, to a lesser extent, of superoxide anion radicals in this cultivar. The H(2)O(2) spots ('burst initiation sites') occurred mainly in the vicinity of leaf veins and correlated in number and distribution with discrete sites of local cell death and with visible symptoms that evolved between 15 and 72 h. The results indicate that ozone effects are amplified in the sensitive tobacco cv. Bel W3 by an oxidative burst which participates in the generation of hypersensitive cell death-like lesions.

Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants.

Hydrogen peroxide (H2O2) has been implicated in many stress conditions. Control of H2O2 levels is complex and dissection of mechanisms generating and relieving H2O2 stress is difficult, particularly in intact plants. We have used transgenic tobacco with approximately 10% wild-type catalase activity to study the role of catalase and effects of H2O2 stress in plants. Catalase-deficient plants showed no visible disorders at low light, but in elevated light rapidly developed white necrotic lesions on the leaves. Lesion formation required photorespiratory activity since damage was prevented under elevated CO2. Accumulation of H2O2 was not detected during leaf necrosis. Alternative H2O2-scavenging mechanisms may have compensated for reduced catalase activity, as shown by increased ascorbate peroxidase and glutathione peroxidase levels. Leaf necrosis correlated with accumulation of oxidized glutathione and a 4-fold decrease in ascorbate, indicating that catalase is critical for maintaining the redox balance during oxidative stress. Such control may not be limited to peroxisomal H2O2 production. Catalase functions as a cellular sink for H2O2, as evidenced by complementation of catalase deficiency by exogenous catalase, and comparison of catalase-deficient and control leaf discs in removing external H2O2. Stress analysis revealed increased susceptibility of catalase-deficient plants to paraquat, salt and ozone, but not to chilling.

The regulation and function of tobacco superoxide dismutases.

Our knowledge of superoxide dismutase (SODs) in tobacco has increased greatly during the past few years. Genes encoding the four identified SOD isoforms of tobacco have been isolated and characterized. Analysis of promoter-beta-glucuronidase fusions has provided information on the cellular expression of SODs in tobacco and has constituted the basis for studying SOD regulation. Constitutive overproduction of SOD has been shown to confer increased tolerance to stress and has started to reveal subtle biochemical differences between SOD isoforms. Thus, thanks to its convenience for molecular and physiological studies, tobacco has come forth in recent years as an excellent model system for studying the regulation and function of SOD in dicotyledonous plants.

Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts.

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inzé [1995] Plant Physiol 107: 737-750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.

Tissue-specific activity of two manganese superoxide dismutase promoters in transgenic tobacco.

In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.

Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts.

Two varieties of tobacco (Nicotiana tabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mn-superoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance in leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. In both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with MV tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that in var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.

Ozone, Sulfur Dioxide, and Ultraviolet B Have Similar Effects on mRNA Accumulation of Antioxidant Genes in Nicotiana plumbaginifolia L.

We have studied the expression of antioxidant genes in response to near ambient conditions of O3, SO2, and ultraviolet B (UV-B) in Nicotiana plumbaginifolia L. The genes analyzed encode four different superoxide dismutases (SODs), three catalases (Cat1, Cat2, and Cat3), the cytosolic ascorbate peroxidase (cyt APx), and glutathione peroxidase (GPx). The experimental setup for each treatment was essentially the same and caused no visible damage, thus allowing direct comparison of the different stress responses. Our data showed that the effects of O3, SO2, and UV-B on the antioxidant genes are very similar, although the response to SO2 is generally less pronounced and delayed. The effects of the different stresses are characterized by a decline in Cat1, a moderate increase in Cat3, and a strong increase in Cat2 and GPx. Remarkably, SODs and cyt APx were not affected. Analysis of SOD and APx expression in the ozone-sensitive Nicotiana tabacum L. cv PBD6 revealed that induction of the cytosolic copper/zinc SOD and cyt APx occurs only with the onset of visible damage. It is proposed that alterations in mRNA levels of catalases and GPx, but not of SODs and cyt APx, form part of the initial antioxidant response to O3, SO2, and UV-B in Nicotiana.

Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants.

Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

Chiral drugs: the FDA perspective on manufacturing and control.

On 27 May 1992, the FDA announced the availability of a policy statement on the development of stereoisomeric drugs. This statement has significant implications for the chemist who is working on the development and validation of analytical controls for chiral drug substances and products. The testing of the bulk drug, the manufacturing of the finished product, the design of stability testing protocols, and the labelling of the drug must all take the chirality of the active ingredient into consideration.

Magnetic resonance imaging measurements and clinical changes accompanying transtentorial and foramen magnum brain herniation.

Current concepts of brain herniation have depended largely on correlating clinical signs and symptoms with indirect radiographic studies and the results of postmortem neuropathology. This article describes measurements on midsagittal magnetic resonance imaging (MRI) scans that distinctly define normal and abnormal rostral-caudal relationships between the diencephalic-mesencephalic junction and the plane of the tentorial incisura, herein termed the incisural line. We similarly provide quantitative MRI scan measurements relating the cerebellum and the plane of the foramen magnum, termed the foramen magnum line. Measurements from 156 midsagittal and 63 coronal MRI scans performed on 123 normal adults, placed the iter of the aqueduct 0.2 +/- 0.8 mm (mean +/- SD) below the incisural line and the cerebellar tonsils 0.1 +/- 2.1 mm below the foramen magnum line. Defining 2 SD from these norms as abnormal, 23 patients with intracranial mass or obstructive lesions showed 4 distinct patterns of brain herniation, i.e., upward or downward transtentorial shift with or without accompanying cerebellar tonsillar herniation. Five patients with posterior fossa masses demonstrated displacement of the iter above the incisura ranging from 1.6 to 6.3 mm. Eighteen patients with supratentorial masses demonstrated displacement of the iter ranging from 2.0 to 11.0 mm below the incisura. Two-thirds of patients with upward and one-half of those with downward transtentorial shift had concurrent tonsillar herniation. In acute illnesses, MRI scan changes anticipated or confirmed clinical signs of brain herniation. In chronic cases, clinical and MRI scans correlated less well, with MRI sometimes revealing major degrees of anatomical herniation well in advance of clinical abnormalities.

Network therapy using videotape disclosures for adult sexual abuse survivors.

Treatment of childhood sexual abuse survivors may be enhanced by a technique designed to generate a therapeutic constituency for the survivor around the disclosure of childhood abuse experiences. The need for validating relationships is hypothesized as a condition for successful treatment. The videotaped disclosure process proceeds in five stages: (1) Deciding to make a tape, (2) making a videotape following a semistructured interview format, (3) the patient viewing the tape, (4) showing the tape to potential therapeutic team members, and (5) possibly using the tape for a confrontation with the abuser. The technique has been used on 27 cases. Case histories are given to illustrate the procedure. Discussion includes potential mechanisms of action and issues in treatment that arise with the use of the method described.

Differential regulation of superoxide dismutases in plants exposed to environmental stress.

Superoxide dismutases (SODs) are metalloproteins that catalyze the dismutation of superoxide radicals to hydrogen peroxide and oxygen. The enzyme is ubiquitous in aerobic organisms where it plays a major role in defense against oxygen radical-mediated toxicity. In plants, environmental adversity often leads to the increased generation of reduced oxygen species and, consequently, SOD has been proposed to be important in plant stress tolerance. Here we describe the isolation of a cDNA clone encoding a cytosolic copper/zinc SOD from Nicotiana plumbaginifolia. Using this, together with previously isolated cDNAs encoding the mitochondrial manganese SOD and the chloroplastic iron SOD as probes in RNA gel blot analyses, we have studied SOD transcript abundance during different stress conditions: in response to light, during photoinhibitory conditions (light combined with high or low temperatures), and in response to a xenobiotic stress imposed by the herbicide paraquat. Evidence is presented that iron SOD mRNA abundance increases whenever there is a chloroplast-localized oxidative stress, similar to the previous finding that manganese SOD responds to mitochondria-localized events. The diverse effects of the different stress conditions on SOD mRNA abundance thus might provide an insight into the way that each treatment affects the different subcellular compartments.

Transient occurrence of extrachromosomal DNA of an Arabidopsis thaliana transposon-like element, Tat1.

Analysis of 11 genomic clones containing the S-adenosylmethionine synthetase 1 gene (sam1) of Arabidopsis thaliana revealed the presence of a 431-base-pair (bp) insertion in the 3' end of sam1 in one of these clones. The inserted sequence, called Tat1, shows structural features of a transposon. It is flanked by a 5-bp duplication of the target site DNA and has 13-bp inverted repeats at its termini. Two highly homologous elements situated in a different genomic context were isolated from a genomic library. Genomic Southern analysis indicates that there are at least four copies of Tat1 present in the A. thaliana ecotype Columbia genome. Different hybridization patterns are observed with DNAs derived from different ecotypes of Arabidopsis thaliana, indicating that the element has moved since the divergence of these ecotypes. In two populations of A. thaliana, linear extrachromosomal Tat1-homologous DNA has been observed. The presented data are consistent with the hypothesis that Tat1 is an active transposable element.