S. Tsuyoshi Ohnishi

A simplified method of quantitating protein using the biuret and phenol reagents

Abstract A new modification of the Lowry method of quantitating protein is introduced, whereby the protein sample is mixed first with a diluted biuret reagent and later with 2 n phenol reagent (undiluted) for color development. The method is superior to the original in (i) extremely stable color development (0.3% change from 20 min to 2 hr), (ii) good reproducibility (±2% for 50–600 μg/ml of protein), (iii) elimination of the need to mix reagents for each assay, (iv) good storability (the diluted biuret reagent is storable for months), (v) simplicity (both reagents are available commercially), and (vi) the biuret method can be immediately converted to the Lowry method if the former does not yield a sufficient absorbance. It was found that the relationship between absorbance and protein concentration is expressed by a straight line with a slope of 1 in the Hill plot.

Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in the rat brain

Forebrain ischemia was produced in the rat by bilateral occlusion of the common carotid arteries combined with hemorrhagic hypotension (30 mmHg). The whole cerebral cortex was homogenized in the presence of the spin trap agent N-tert-butyl-alpha-phenyl-nitrone, followed by a Folch extract. Spin-adducts were detected using electron spin resonance spectroscopy. The lipid peroxidation was estimated from both the amount of thiobarbituric acid reactive substance and the formation of conjugated diene. After 10 or 20 min of ischemia, reperfusion was initiated which induced an abrupt burst of free radical formation. The formation peaked at 5 min, and the peak value increased with the ischemia time. The degree of lipid peroxidation, which was measured after 20 min of reperfusion, also increased with the ischemia time. The results suggest that the lipid peroxidation may be the direct consequence of the action of free radicals formed during ischemia and reperfusion periods.

Calcium-induced Ca2+ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: The effects of halothane and dantrolene

Calcium‐induced calcium release and halothane‐induced calcium release from pig sarcoplasmic reticulum (SR) were studied. The SR prepared from pig susceptible to malignant hyperthermia (MH) was shown to release calcium at a much lower level of calcium content than in normal pig SR. The concentration above which halothane can release calcium is 40 μM for both MH‐SR and normal SR, although the latter required a high calcium content to demonstrate the calcium release. Dantrolene was shown to inhibit the halothane‐induced calcium release. Results suggests that SR plays an importnat role in pathogenesis of MH.

Attenuation of rat ischemic brain damage by aged garlic extracts: a possible protecting mechanism as antioxidants.

Effects of an aged garlic extract and its thioallyl components on rat brain ischemia were examined using a middle cerebral artery occlusion model and a transient global ischemia model. In focal ischemia, an aged garlic extract, S-allyl cysteine (SAC), Allyl sulfide (AS) or Allyl disulfide (ADS) was administered 30 min prior to ischemic insult. Three days after ischemic insult, water contents of both ischemic and contralateral hemispheres were measured to assess the degree of ischemic damage. The water content of the ischemic control (no drug treatment) group was 81.50 +/- 0.07% (mean +/- SEM). It was significantly reduced with the administration of 300 mg/kg of SAC; the water content was 80.66 +/- 0.11% (P < 0.001). The histological observation using 2,3,5-triphenyltetrazolium chloride staining demonstrated that the administration of SAC reduced infarct volume. Neither AS nor ADS was effective. In global ischemia, the production of reactive oxygen species (ROS) was measured ex vivo using a spin-trapping agent, alpha-phenyl-N-tert-butylnitrone, and electron paramagnetic resonance spectroscopy. The production of ROS had two peaks; first at 5 min and second at 20 min after reperfusion. Both SAC and 7-nitro indazole, a nitric oxide synthase inhibitor, did not attenuate the amount of ROS produced at the first peak, but did the amount of the second peak. A possible involvement of peroxinitrite, which may be formed from superoxide and nitric oxide and is known to be highly toxic in ischemia/reperfusion injury of the brain, was suggested.

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles.

The time-course of Ca2+ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Several methods were used to trigger Ca2+ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca2+ concentration ([Ca2+0]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca2+ release induced by either halothane or Ca2+ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca2+ released by halothane at low [Ca2+0] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca2+ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca2+ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum (k = 83 s-1) than in normal (k = 37 s-1). Thus, all types of Ca2+ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca2+ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.

S-Allylcysteine Inhibits Free Radical Production, Lipid Peroxidation and Neuronal Damage in Rat Brain Ischemia

The efficacy of S-allylcysteine (SAC) as a free radical scavenger was studied using rat brain ischemia models. In a middle cerebral artery occlusion model, preischemic administration of SAC had the following effects: it improved motor performance and memory impairment and reduced water content and the infarct size. In a transient global ischemia model, the time course of free radical (alkoxyl radical) formation as studied by electron paramagnetic resonance (EPR) spectroscopy and alpha-phenyl-N-tert-butylnitrone (PBN) was biphasic; the first peak occurred at 5 min and the second at 20 min after reperfusion. Although SAC did not attenuate the first peak, it did affect the second peak, which is related to lipid peroxidation. The lipid peroxidation as estimated by thiobarbituric acid reactive substances (TBARS) increased significantly at 20 min after reperfusion. SAC decreased TBARS to the levels found without ischemia. These results suggest that SAC could have beneficial effects in brain ischemia and that the major protective mechanism may be the inhibition of free radical-mediated lipid peroxidation.

Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca++-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO2 = 21%, n = 5) or hypoxic (FiO2 = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO4.7H2O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca2+- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.

Electron Paramagnetic Resonance (EPR) Detection of Nitric Oxide Produced during Forebrain Ischemia of the Rat

To detect if nitric oxide (NO) is produced in rat forebrain ischemia, we applied an electron paramagnetic resonance (EPR) NO-trapping technique. We also performed a detailed characterization of the technique. Diethyldithiocarbamate (DETC) and Fe-citrate were used as NO-trapping reagents. Under controlled ventilation, forebrain ischemia was produced by occlusion of both carotid arteries combined with hemorrhagic hypotension at 50 mm Hg for 15 min. DETC and Fe were administered 30 min prior to the onset of ischemia. During ischemia, the cerebral cortex was removed, and EPR samples were prepared. At liquid nitrogen temperatures, the NO-Fe-DETC signal (a triplet signal centered at g = 2.039 with the hyperfine coupling constant aN of 13 G) was detected overlapping Cu-DETC signals. By perfusing various concentrations of an NO-generating agent, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, into the rat brains, the amount of the “trapped NO” was calibrated. The size of the NO-Fe-DETC signal was well correlated with the NO concentrations in the perfusate (correlation coefficient r = 0.998, p < 0.01). Based on this calibration curve, it was found that the amount of trapped NO during forebrain ischemia increased to seven times that of the control (control n = 5, forebrain ischemia n = 4, p < 0.005).

Electron paramagnetic resonance study on nitric oxide production during brain focal ischemia and reperfusion in the rat

The production of nitric oxide (NO) during brain focal ischemia and reperfusion was measured using diethyldithiocarbamate (DETC)/Fe-citrate, NO trapping reagents, and electron paramagnetic resonance spectroscopy. The NO production is potentiated after 5 min of ischemia, and is continued during 60 min of ischemia. During the reperfusion period after 60 min of ischemia, NO was also produced. However, its production during reperfusion was not observed when the ischemia time was less than 15 min. The NO signal during reperfusion after 60 min of ischemia decreased after 15 min of reperfusion. These results suggest that NO production during ischemia is a physiological reaction for increasing cerebral blood flow, while NO production during reperfusion may be related to cellular damage.

Three-Dimensional Imaging of Nitric Oxide Production in the Rat Brain Subjected to Ischemia—Hypoxia

By the systemic administration of diethyldithiocarbamate and iron into the rat, nitric oxide radicals produced in the brain during ischemia–hypoxia were trapped. The right hemisphere of the brain was then removed and frozen with liquid nitrogen. With use of recently developed electron paramagnetic resonance imaging instrumentation and techniques, three-dimensional imaging of the production of the nitric oxide radicals in several brains was performed. The results suggest that nitric oxide radicals were produced and trapped in the areas that are known to have high nitric oxide synthase activity, such as cortex, hippocampus, hypothalamus, amygdala, and substantia nigra. In this ischemia–hypoxia model, which did not interrupt the posterior circulation, the production and trapping of nitric oxide in the cerebellum were ∼30% of those in the cerebrum.