Hormonal regulation of gene expression in barley aleurone layers

As part of our investigation of the mode of action of plant hormones in barley (Hordeum vulgare L.) aleurone layers, we have studied the expression of five identified and three unidentified mRNA species in the presence of exogenous gibberellic acid (GA₃) and abscisic acid. Three of the mRNAs are GA₃-inducible, three are suppressed by GA₃, and two are constitutive. The extent of the GA₃ effect differs considerably for both inducible and suppressible mRNAs. For example, a ten-fold higher concentration of GA₃ (10^-8 M) is required for full induction of the high-pl group -amylase mRNA than is required for the low-pI -amylase mRNA (10^-9 M). Temporal regulation of mRNA abundance also varies between the two -amylase isoenzyme groups. The three GA₃-suppressible mRNA species studied, alcohol dehydrogenase (ADH1), a probable amylase and protease inhibitor, and an unidentified barley mRNA species also varied in response to GA₃. The ADH1 mRNA decreased drastically within 8 h of GA₃ treatment, whereas the other two began to decrease in abundance only after 12–16 h of GA₃ treatment. Abscisic-acid treatment counteracted the GA₃ effects for both the inducible and suppressible mRNA species. Comparison of -amylase-mRNA levels and -amylase-synthesis rates showed a strong correlation between the two parameters, the only exception being a lack of -amylase synthesis in the presence of -amylase mRNA at low GA₃ concentrations. Therefore, the expression of -amylase seems to be regulated primarily by its mRNA levels.Abbreviations ABA abscisic acid - ADH1 alcohol dehydrogenase 1 - cDNA copy DNA - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor     

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate `leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.     

Endurance training in dogs increases vascular responsiveness to an alpha 1-agonist

The effects of endurance training on vascular responsiveness to an alpha 1-agonist and the associated changes in baroreflex modulation of heart rate and vascular resistance were studied. Graded dosages of phenylephrine were given to eight treadmill-trained dogs and to eight untrained dogs; both groups were chronically instrumented and were sedated and resting when tested. These dosages were repeated after ganglionic blockade. Aortic pressure, cardiac output, central venous pressure, peripheral resistance, and heart rate were each averaged over 30 s before injection and 90 s after injection. The slope of the peripheral resistance-dose relationship was significantly increased in trained compared with untrained dogs in both the unblocked and blocked cases [unblocked: trained 0.89, untrained 0.47; blocked: trained 4.30, untrained 2.05 (mmHg.l-1.min)/(microgram.kg-1)]. The unblocked resistance slopes were reduced with respect to the blocked slopes by 77 (untrained) and 79% (trained). The slope of the heart rate-aortic pressure response was reduced, but not significantly, by endurance training. We conclude that 6 wk of endurance training in dogs resulted in a doubling of the vascular responsiveness to an alpha 1-agonist, with no significant change in the baroreflex regulation of resistance or heart rate.     

Resistance ofSpodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory

Median lethal doses (LD₅₀s) of nuclear polyhedrosis virus (NPV) were determined in neonatal offspring ofSpodoptera frugiperda (J. E. Smith) (Sf) larvae captured in southeastern Louisiana in 1981, 1982, and 1984. These LD₅₀s ranged from 1.8 to 16.3 polyhedral inclusion bodies (PIB)/insect. The LD₅₀s significantly (P<0.05) increased during the season of 1982 but had no pattern in 1981 or 1984. However, the Sf populations increased in heterogeneity of response to the NPV during all 3 years. The LD₅₀ increased from 4.1 to 18.7 PIB/insect in a Sf laboratory colony exposed to the NPV LD₈₀ for 7 generations, whereas in a control colony not exposed to NPV the LD₅₀ was 5.9 PIB/insect after 7 generations.     

Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice

Summary Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Spinach leaf acetyl-coenzyme a synthetase: purification and characterization

Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M_r value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K_i for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA.     

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.