Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

Characteristic Amino Acid Sequences of Chloroplast and Cytosol Isozymes of CuZn-Superoxide Dismutase in Spinach, Rice and Horsetail

Two isozymes of CuZn-superoxide dismutase (SOD) were purifiedfrom spinach. One (CuZn-SOD II) was localized in chloroplastsand had the same properties as the enzyme previously reported[Asada et al. (1973) Eur. J. Biochem. 36: 257–266]. Theother isozyme (CuZn-SOD I) was predominantly expressed in seedsand in etiolated seedlings of spinach, but was localized inthe cytosol of the leaves as a minor enzyme. The isozymes havesimilar molecular weights, subunit structures, and metal contents;but their amino acid compositions, absorption spectra, CD spectraand sensitivities to hydrogen peroxide are different. The amino acid sequences of 50 amino-terminal residues of thechloroplast and cytosol isozymes of CuZn-SOD from spinach, riceand horsetail were determined and compared with those of CuZn-SODsfrom other plants. The sequences can be divided into chloroplastand cytosol types, and characteristic sequences can be identifiedin accordance with the observations that the two types of CuZn-SODisozymes from green algae, ferns and angiosperms can be distinguishedimmunologically from each other. Differences in amino acid sequencesamong the cytosol enzymes are greater than those among the chloroplastenzymes, indicating that the rate of mutation of the cytosolCuZn-SOD is higher than that of the chloroplast CuZn-SOD. Theseresults provide further evidence that the divergence of thetwo types of isozyme of CuZn-SOD occurred at a very early stageof its acquisition, and that each type of CuZn-SOD has evolvedindependently. (Received September 1, 1989; Accepted November 6, 1989)     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Specificity of Pseudomonas Isoamylase

The cell-free extract of Aspergillus niger (AKU 3302) catalyzed the degradation of nicotinamide mononucleotide to an unidentified compound. This compound was isolated from the reaction mixture and identified as nicotinic acid ribonucleoside (NAR). When the mold was cultured on media containing nicotinic acid- and nicotinamide-¹⁴C, the ratio of radioactivity incorporated into NAR was 16 and 10% of the total activity in the mycelial extracts, respectively.     

Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids

Thylakoid-bound and stromal ascorbate peroxidases scavenge thehydrogen peroxide that is photoproduced in PSI of chloroplastthylakoids. The primary oxidation product of ascorbate in thereaction catalyzed by ascorbate peroxidase, the monodehydroascorbate(MDA) radical, is photoreduced by thylakoids [Miyake and Asada(1992) Plant Cell Physiol. 33: 541]. We have now shown thatthe photoreduction of MDA radical in spinach thylakoids is largelydependent on ferredoxin (Fd), as determined by the monitoringthe MDA radical by electron paramagnetic resonance. Further,the reduced Fd generated by NADPH and Fd-NADP reductase couldreduce the MDA radical at a rate of over 10⁶ M^–1 s^–1,indicating that the photoreduced Fd in PSI directly reducesthe MDA radical to ascorbate. Photoreduction of NADP⁺ by spinach thylakoids was suppressedby the MDA radical and conversely that of MDA radical was suppressedby NADP⁺, indicating a competition between the MDA radical andNADP⁺ for the photoreduced Fd in PSI. The ratio of the rateconstant for the photoreduction of MDA radical to that for thephotoreduction of NADP⁺ was estimated to be more than 30 to1. Thus, MDA radical is preferentially photoreduced as comparedto NADP⁺. From these results, we propose that the thylakoid-boundascorbate peroxidase and the Fd-dependent photoreduction ofMDA radical in PSI are the primary system for the scavengingof the hydrogen peroxide that is photoproduced in the thylakoids. (Received December 9, 1993; Accepted February 16, 1994)     

The advancement in the early diagnostics of developmental hip dysplasia in infants--the role of ultrasound screening

Developmental hip dysplasia (DDH) is a deformation of the locomotor system that can occur on previously normal bone structures in late fetal development. The study was undertaken to determine the value of continuous ultrasound screening in early diagnosis of DDH. In the area covered by the Labin Primary Health Center, clinical and ultrasound examination of the hips was performed in all children during the fourth month of life from 1989 to 2001. The incidence of DDH was 3.3%. The authors compared the obtained results with results of clinically examined hips in the period from 1968 to 1988 (incidence 1.7%), before the ultrasound diagnostics was introduced. The hip radiograms were performed only in children with clinically positive signs of higher degree of DHD. The ultrasound examination was performed with Hitachi EUB 410 real time linear transducer with a 5 MHz probe. The examiners used methods and criteria suggested by Graf. The most useful feature of the ultrasound method is that it allows the visualization of cartilaginous femoral head and its relationship to the acetabulum in infants. The method is non-invasive, repeatable, without risk of ionizing radiation or need for the contrast material administration. The results of the study revealed an important increase of DHD incidence after the introduction of ultrasound examination. US is highly sensitive in hip imaging and reveals the existence of a number of dislocated and subluxated hips that otherwise would be missed. The anomaly was discovered in the early period of life, which is very important for appropriate therapeutic effects.     

Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts and Photoreduction of Its Primary Oxidation Product Monodehydroascorbate Radicals in Thylakoids

Ascorbate peroxidase, a key enzyme for the scavenging of hydrogenperoxide in chloroplasts, was found in a thylakoid-bound formin spinach chloroplasts at comparable activity to that in thestroma. The activity of peroxidase was detectable in the thylakoidsonly when prepared by an ascorbate-containing medium, and enrichedin the stroma thylakoids. The thylakoid enzyme was not releasedfrom the membranes by either 2 mM EDTA, 1 M KCl, 2 M NaBr or2 M NaSCN, but was solubilized by detergents. Enzymatic propertiesof the thylakoid-bound ascorbate peroxidase were very similarto those of the stromal ascorbate peroxidase. Thylakoid-bound ascorbate peroxidase could scavenge the hydrogenperoxide either added or photoproduced by the thylakoids. Nophotoreduction of hydrogen peroxide was observed, however, inthe thylakoids whose ascorbate peroxidase was inhibited by KCNand thiol reagents or inactivated by the treatment with ascorbate-depletion.The primary oxidation product of ascorbate in a reaction ofascorbate peroxidase, monodehydroascorbate (MDA) radical, wasphotoreduced in the thylakoids, as detected by the quenchingof chlorophyll fluorescence, disappearance of EPR signals ofthe MDA radicals and the MDA radical-induced oxygen evolution.Thus, ascorbate is photoregenerated in the thylakoids from theMDA radicals produced in a reaction of ascorbate peroxidasefor the scavenging of hydrogen peroxide. (Received March 26, 1992; Accepted April 22, 1992)     

Changes in organelle superoxide dismutase isoenzymes during air adaptation of submerged rice seedlings: Differential behaviour of isoenzymes in plastids and mitochondria

Changes in activities and levels of superoxide dismutase (SOD, EC 1.15.1.1) isoenzymes were studied during air adaptation of submerged rice (Oryza sativa L.) seedlings. Seeds were germinated for 6 d in the dark under water (submerged), and then for another day in air (air-adapted). For a control, seeds were germinated for 6 d throughout in air (aerobic). Staining for activity of SOD of shoot extracts showed a total of five major distinct forms of SOD: one mitochondrial Mn enzyme (mtSOD) and four CuZn enzymes, one of which was plastidic (plSOD) and the other three cytosolic (cytSOD). Activity of plSOD was much lower in submerged seedlings than in aerobic controls and increased after exposure to air. In contrast, mtSOD activity in submerged seedlings was as high as that found in aerobic controls, and did not increase upon exposure to air. One of the cytSODs showed responses similar to those of plSOD. The activities of another two cytSODs were slightly lower in submerged seedlings than those in aerobic controls, but decreased after 24 h of air adaptation. Western blot analysis revealed that these changes in activities of SODs are due to changes in the levels of their enzyme proteins. We also followed changes in the levels of cytochrome c and ferredoxin-NADP⁺ reductase (EC 1.6.99.4) as indices of the development of mitochondria and plastids, respectively. Organelle SODs were always present at higher levels than would be expected in view of the development of the electron-transport systems of the corresponding organelles during submergence and the subsequent air-adaptation period.Abbreviations cytSOD cytosolic CuZn-superoxide dismutase - FNR ferredoxin-NADP⁺ reductase - mtSOD mitochondrial Mn-superoxide dismutase - plSOD plastidic CuZn-superoxide dismutase - SOD superoxide dismutase We gratefully acknowledge Dr. A. Wada, Kyoto University, for the use of a densitometer for this work, and also would like to thank Y. Maki, Kyoto University, for help with the densitometry of the photographs.     

The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes

The photoreduction of dioxygen in spinach thylakoid membraneswas enhanced about 10-fold by the FAD-enzyme monodehydroascorbateradical (MDA) reductase at 1µM. The primary photoreducedproduct of dioxygen catalyzed by MDA reductase was the superoxideradical, as evidenced by the inhibition of photoreduction ofCytc by superoxide dismutase. The apparent K_m for dioxygen ofthe MDA reductase-dependent photoreduction of dioxygen was 100µM,higher by one order of magnitude than that observed with thylakoidmembranes only. Glutathione reductase, ferredoxin-NADP⁺ reductase,and glycolate oxi-dase also mediated the photoproduction ofsuperoxide radicals in thylakoid membranes at rates similarto those with MDA reductase. Among these flavoenzymes, MDA reductaseis the most likely mediator stimulating the photoreduction ofdioxygen in chloroplasts; its function in the protection fromphotoinhibition under excess light is discussed. (Received February 24, 1998; Accepted May 19, 1998)     

Long-Term Effect of Different Physical Activity Levels on Subclinical Atherosclerosis in Middle-Aged Men: A 25-Year Prospective Study

Background

The purpose of the study was to investigate the influence of lifetime physical activity (PA) on selected indices of atherosclerosis in longitudinal observation of middle-aged men.

Methods

The subject of the study was a cohort of 101 men (mean age 59,7±9,0 years), free of cardiovascular symptoms and treatment, participating in follow-up examinations in the years 1985/90-2011/12. Self-report PA was assessed by interviewer-administered Seven-Day PA Recall and Historical PA questionnaire. Subclinical atherosclerosis was measured by assessing the coronary artery calcification (CAC) according to Agatston''s method using multi-slice computed tomography; the carotid intima-media thickness (IMT) using high-resolution B-mode ultrasound; and the reactive hyperemia index (RHI) using peripheral arterial tonometry (EndoPAT2000). The participants were initially divided into three groups according to tertiles of exercise-related energy expenditure (EE) in kcal/week at baseline, i.e. <2050 (low-to-moderate; n = 33), 2050–3840 (high; n = 34), >3840 (very high; n = 34).

Results

The low-to-moderate, high and very high PA groups were comparable in terms of age and atherosclerosis risk factors at baseline. No linear relationship was found between PA and CAC, IMT and RHI. Men who maintained low-to-moderate (n = 26), high (n = 21) and very high (n = 15) PA level had the mean CAC of 286.1±361.9, 10.7±28.9, and 106.1±278.3 (p<0.001 for low-to moderate vs high; p<0.05 for low-to-moderate vs very high); the mean IMT of 0.751±0.19 mm, 0,641±0.26 mm, and 0.750±0.60 mm (p>0.05); and the mean RHI of 1.69±0.4, 2.00±0.4, and 2.13±0.5 (p for trend = 0.050), respectively. No cases of CAC>400, IMT ≥0.9 and RHI<1.67 were noted only among men with maintained high PA level. At final examination men with high and very high PA had more favorable cardiometabolic profile than men with lower PA.

Conclusions

Maintaining regular high PA level through young and middle adulthood may protect against atherosclerosis as measured by CAC, IMT and RHI.