Free radical generation coupled with arachidonate lipoxygenase reaction relates to reoxygenation induced myocardial cell injury.

OBJECTIVE: The role of arachidonate lipoxygenase activity in reoxygenation induced cell injury in adult canine cardiac myocytes was investigated. METHODS: The production of hydroxyeicosatetraenoic acids (HETEs), which are lipoxygenase metabolites, was measured with high pressure liquid chromatography in canine cardiac myocytes cultured under hypoxic conditions and then reoxygenated. Free radical generation was evaluated by electron paramagnetic resonance spectroscopy with a spin trapper, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and luminol enhanced chemiluminescence emission. Cell injury was estimated in terms of morphological changes and release of intracellular enzymes. Morphological damage to myocytes was quantified in terms of the percentage of hypercontracted "round" cells. The effects of nordihydroguaiaretic acid, AA-861, mepacrine, indomethacin, aspirin, alpha tocopherol, and 2-0-octadecylascorbic acid (CV-3611) on lipoxygenase metabolism, free radical generation and cell injury were also assessed. RESULTS: Cardiac myocytes produced 5-HETE and 12-HETE at less than 0.1 ng.mg-1 protein under normoxic conditions. Production of HETE was greatly increased at five hours of reoxygenation after 45 minutes of hypoxia [5-HETE = 12.0(SEM 0.5), 12-HETE = 23.6(1.1) ng.mg-1 protein]. Both DMPO-OH adduct generation and chemiluminescence emission were considerably increased after one to three hours of reoxygenation, although they increased only slightly after 45 minutes of hypoxia. After five hours of reoxygenation, long rod cells gradually became deformed; 92.0% of the cells were converted to hypercontracted "round" cells. Cell injury and HETE production were significantly suppressed by nordihydroguaiaretic acid (10 microM), AA-861 (2 microM), and mepacrine (10 microM). Indomethacin (10 microM) and aspirin (50 microM) enhanced cell injury and HETE production. alpha Tocopherol and CV-3611 greatly suppressed cell injury and free radical generation, but not HETE production. CONCLUSION: The arachidonate lipoxygenase metabolic pathway may have an important role in reoxygenation induced myocardial cell injury in adult cardiac myocytes, possibly because of the generation of free radicals.

Adenosine blockade during reperfusion reverses the infarct limiting effect in preconditioned canine hearts.

OBJECTIVES: The aim was to investigate whether adenosine release after reperfusion contributes to infarct limitation by ischaemic preconditioning. METHODS: Dogs underwent preconditioning with four 5 min cycles of left anterior descending coronary artery (LAD) occlusion and reperfusion, followed by 90 min LAD occlusion and 5 h reperfusion with or without the non-specific adenosine receptor blocker, 8-phenyltheophylline (8-PT). Infarct size was assessed by a dual staining method with triphenyltetrazolium chloride and Evans blue. Blood flow measurements in the subendocardial region were made by infusion of coloured microspheres before occlusion and midway through the sustained occlusion. Transcardiac alteration of neutrophillic function was assessed by luminol-enhanced whole blood chemiluminescence induced by zymosan. RESULTS: Infarct size was significantly reduced in the preconditioned dogs [12.5(SEM 4.0)%, n = 10, p < 0.01] compared with the control dogs [40.5(6.1)%, n = 10], an effect significantly reduced by the 8-PT treatment [23.4(4.9)%, n = 8]. Treatment with 8-PT without ischaemic preconditioning had no effect on infarct size [42.8(6.3)%, n = 7]. There was no difference in myocardial blood flow in the ischaemic or non-ischaemic subendocardial tissue between any pair of the four groups. The ratio of whole blood chemiluminescence in the cardiac vein to that in the carotid artery was considerably reduced in preconditioned dogs compared with that in control dogs after reperfusion. Myeloperoxidase activity in the ischaemic myocardium and the peripheral neutrophil count at the end of the experiment were both also decreased compared with control dogs. In preconditioned dogs treated with 8-PT, neutrophillic function in the coronary circulation after reperfusion was increased compared with that in both controls and preconditioned dogs with no 8-PT treatment. There was no difference in neutrophillic function between the 8-PT-treated dogs with or without ischaemic preconditioning. Treatment with 8-PT increased myeloperoxidase activity in the ischaemic myocardium of the preconditioned dogs, and no difference was seen in activity between dogs treated with 8-PT with or without ischaemic preconditioning. CONCLUSIONS: An adenosine receptor blocker caused a moderate but significant reversal of infarct limitation by ischaemic preconditioning associated with a significant increase of neutrophillic function in the coronary circulation during early reperfusion.

Transcardiac alteration of neutrophil function relates to myocardial ischaemia/reperfusion injury.

OBJECTIVE: The aim was to define the relation between transcardiac changes in neutrophil function in myocardial ischaemia and the progression of myocardial necrosis. METHODS: Samples of blood from carotid artery, jugular vein, and cardiac vein streaming from the ischaemic area were taken simultaneously in a canine coronary occlusion-reperfusion model of myocardial infarction. Neutrophil function was evaluated by neutrophil count, whole blood chemiluminescence and leucocyte infiltration into the ischaemic myocardium. Myocardial necrosis was assessed by plasma creatine kinase and dual staining technique using Evans blue dye and triphenyltetrazolium chloride. Effects of a free radical scavenger, N-2-mercaptopropionyl glycine (MPG), initiated 15 min before reperfusion and continued during the reperfusion phase, were also examined. RESULTS: Whole blood chemiluminescence of the cardiac vein was reduced at 90 min after coronary artery occlusion as compared to carotid artery [5.8(SEM 0.5) v 7.5(0.7) count x 10(3) cell neutrophil-1 x 10 min-1, p < 0.05], and then increased abruptly after reperfusion to peak after 10 min of reperfusion [7.1(0.7) count x 10(3) cell neutrophil-1 x 10 min-1]. The neutrophil count in cardiac venous blood was significantly reduced within 5 min of reperfusion. MPG significantly attenuated the reperfusion associated increase in cardiac vein whole blood chemiluminescence and the decrease in the cardiac venous blood neutrophil count. The increase in myocardial free radical generation 1-3 h after reperfusion, as assessed by the electron paramagnetic resonance spin trapping technique, was reduced markedly, as was the extent of leucocyte infiltration into the ischaemic myocardium. Under these conditions, administration of MPG significantly reduced myocardial infarct size [40.3(4.5)% v 21.4(4.2)%, p < 0.05]. A marked increase in transcardiac creatine kinase release after reperfusion observed in control dogs was also reduced significantly. CONCLUSIONS: A transient alteration of neutrophil function occurs in the coronary circulation immediately after reperfusion, which may augment neutrophil infiltration and free radical generation in the ischaemic myocardium, leading to the propagation of myocardial ischaemia/reperfusion injury.

Amino-terminal and carboxyl-terminal half-molecules of ovotransferrin: preparation by a novel procedure and their interactions.

The amino- (N-) and carboxyl- (C-)terminal half-molecules of ovotransferrin were prepared by a novel procedure. The trypsin-nicked ovotransferrin (Ikeda et al. (1985) FEBS Lett. 182, 305-309), in which the two half-molecules interact non-covalently forming a stable dimer, was purified by gel filtration and anion-exchange column chromatography. By subsequent cation-exchange chromatography, the nicked form was distinctly separated into an equivalent amount of the N-terminal and C-terminal half-molecules. Analyses of the N-terminal and C-terminal sequences indicated that the N-terminal and C-terminal half-molecules comprised the alignments of residues 1-332 and 342-686 of ovotransferrin, respectively. Anion-exchange chromatography, gel filtration chromatography, and non-denaturing polyacrylamide gel electrophoresis revealed that the isolated half-molecules had the ability to re-associate in solution. The contents of alpha-helix and beta-sheet of the two half-molecules, as determined by circular dichroism (CD) spectra, were very similar to those of intact ovotransferrin. No prominent alteration in the secondary structure of the two half-molecules was induced by the re-association.

Effects of anion binding on the conformations of the two domains of ovotransferrin.

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.

Changes in directed attention and short-term memory in depression.

This study examined changes in directed attention and short-term memory in depression using a newly constructed battery of computerized measures. A repeated measures design was used with two sample groups; 25 individuals meeting DSM-IV criteria for Major Depression and a group-matched comparison sample of 27. Both groups were tested at three points in time over a 10-week period. Test-retest reliability of the measures was examined. Profile analysis demonstrated that there were differences between the depressed and comparison groups in both directed attention and short-term memory. Recommendations for specific improvements in the testing battery are discussed. The ability to detect changes in directed attention and short-term memory may have clinical utility in early detection of impending onset of depression or subtle residual symptoms of an acute episode that may impair functioning or signal a relapse.

Inhibitory pathway of cardiac PKA-dependent Cl- conductance via pertussis toxin-sensitive G proteins.

The inhibitory pathway of cardiac cAMP-dependent protein kinase regulated Cl- conductance was investigated using the whole-cell configuration of patch-clamp techniques in single guinea pig ventricular myocytes. Pertussis toxin-sensitive G proteins (Gi), mediating the signal transductions between muscarinic receptors and adenylate cyclase, have a substantial tonic activity even in the absence of muscarinic receptor modulators. Muscarinic agonists or antagonists (like atropine) either increase or decrease this basal activity of Gi by altering the proportion of active and inactive forms of the receptors. Similar to L-type Ca-channel currents, the Cl- conductance showed a transient over-recovery upon cessation of brief muscarinic receptor stimulation by carbachol (CCh) (rebound). Atropine alone enhanced the Cl- conductance elicited by low concentrations of Iso (reverse agonist). After washout of atropine, the over-suppression of the conductance was observed as a mirror image of CCh-induced rebound (reverse rebound). Both types of rebound became prominent when cell dialysis with pipette solutions containing 100 microM GTP was minimized with high-resistance pipettes. Endogenous GTP is therefore an intracellular modulator, and not simply a mediator, of Gi-dependent signal transduction.

A random controlled study of the prophylactic effect of lysozyme chloride on post-transfusion hepatitis.

In a study designed to determine the preventive effect of lysozyme on post-transfusion hepatitis, 260 patients with orthopedic diseases, who received blood by transfusion during a period of 5 years from June 1970 to May 1975, were divided at random into a group of 123 patients treated with lysozyme is doses of 60 to 170 mg/day for 4 to 24 weeks, and a control group of 137 patients. The incidence of post-transfusion hepatitis was 10 (8.1%) out of 123 patients for the treated group, and 28 (20.4%) out of 137 patients for the control group. Icteric hepatitis occurred in 1 (0.8%) patient in the treated group, while its incidence in the control group was 8 (5.8%) out of 137 patients.

Visualisation method of spatial interictal discharges in temporal epilepsy patients using magneto-encephalogram.

The aim of the study was to develop a method for investigating how interictal epileptic discharges in temporal epilepsy patients are activated spatially. The activity was measured using magneto-encephalography (MEG). The MEG data were used to produce a current-arrow map that reflected the topographic distribution of the electrical current for each peak epileptic waveform. A large current distribution was obtained that appeared to be contained in the limbic structure, in each temporal lobe. The large current orientation indicated two opposite directions. Furthermore, the decrease in the maximum strength of the current-arrow, depending on the medication (e.g. the decrease from 11 to 6 pT m-1 in the left temporal lobe (contralateral stimuli)), suggested that the discharge distributions could be used to verify the efficacy of medication. Thus the topographical visualisation method could be a new strategy for diagnosis in temporal epilepsy patients.

Radioassay of the folate-hydrolyzing enzyme activity, and the distribution of the enzyme in biological cells and tissues.

A sensitive radioassay method has been developed to quantitate the activity of the folate-hydrolyzing enzyme which catalyzes the hydrolysis of folic acid to pteroic acid and glutamic acid. The method is based on analyzing [2-14C]pteroic acid separated by a thin-layer chromatography on an Avicel SF cellulose plate using 0.1 M potassium phosphate buffer, pH 7.0, as a solvent. This method was found to be more sensitive than a conventional photometric method to determine the activity of the folate-hydrolyzing enzyme. High activities of the enzyme were found in Crithidia fasciculata ATCC 12857, Neurospora crassa IFO 6979 and rat liver. Smaller activities of the enzyme were widely distributed in other microbial cells and mammalian tissues.

[Neuroimaging findings of hemiconvulsions, hemiplegia, epilepsy (HHE) syndrome].

A 32-year-old man with hemiconvulsions, hemiplegia, epilepsy (HHE) syndrome is described. He was well developed with a normal pregnancy and delivery, but at age 10 months, he had status epilepticus during a febrile illness. Thereafter, he was noted to have left hemiparesis and mental retardation with recurrent hemiconvulsions. Magnetic resonance (MR) images showed atrophy and degeneration of the right cerebral cortex and white matter, homolateral thalamus, caudate nucleus, and hippocampus, with hyperintensities in both T2-weighted (TR/2200, TE/90) and proton (TR/2200, TE/30) images. There were also slight bilateral cerebellar atrophies. Quantitative single photon emission computed tomographic (SPECT) images using technetium-99m-ethyl cysteinate dimer (99mTc-ECD) revealed markedly reduced cerebral blood flow (CBF) in the right cerebral hemisphere, homolateral thalamus, caudate nucleus and bilateral cerebellum. Bilateral putamen and the medial occipital lobe showed normal findings on MR images and normal regional CBF in SPECT images. We suppose these selective neuronal injures in this case of HHE syndrome will be mainly due to histotoxic factors in epileptic brain damage.

[A case of progressive continuous muscular rigidity and painless and rhythmic muscle spasm associated with autoantibody against glutamic acid decarboxylase].

We described a 60-year-old man with 5-year history of insulin dependent diabetes mellitus who developed continuous rigidity of truncal muscle and painless, rhythmic muscular spasm of trunk and proximal lower and upper extremities. The rigidity continued even in sleep. The painless muscle spasm was often precipitated by volitional movement and emotional stimuli. Intravenous administration of diazepam strongly attenuated the muscle spasm as well as truncal rigidity. Surface electromyography showed the continuous contraction of abdominal and paraspinal muscles. The rhythmic, clonic spasm of shoulder, triceps brachii, intercostal, abdominal, paraspinal and quadriceps femoris muscle induced by voluntary neck flexion was not compatible with typical stiff-man syndrome. Antibody against glutamic acid decarboxylase (GAD) was detected in the serum and cerebrospinal fluid of this patient. His condition was getting well with oral intake of sodium valproate. While painless, rhythmic spasm and persistent rigidity during sleep ruled out the patient from typical stiff-man syndrome, he was supposed to have the same pathophysiological mechanism as the anti-GAD autoantibody positive stiff-man syndrome.

Predictor of cognitive deterioration in elderly subjects by auditory-evoked magnetic signal using magnetoencephalography.

Recent development of auditory-evoked magnetoencephalography (A-MEG) made it possible to measure interhemispheric neural conduction time (INCT) of auditory impulses. We estimated INCT with A-MEG and cognitive function with mini-mental state examination (MMSE) in 85 elderly patients with chronic dizziness (CD) and found that INCT was negatively correlated with MMSE scores (p<0.001). In 11 of 85 patients whose MMSE scores were within the normal range, A-MEG and MMSE were repeated for the subsequent 4 years to find longitudinal changes in INCT and cognitive function. The 11 patients were divided into two groups according to the baseline INCT values, such as Group A with normal INCT (n=7) and Group B with abnormally prolonged INCT (n=4). In Group A, INCT and MMSE scores remained within the normal range throughout the 4-year period. In Group B, INCT showed the tendency towards progressive prolongation during the follow-up period, and MMSE scores decreased to abnormally low levels at the third or fourth follow-up year in all the patients. The present results suggest that rapid neural interaction of both cerebral hemispheres is needed to maintain normal cognitive function. Abnormal INCT prolongation in elderly subjects suggests subclinical cortical network dysfunction and may predict the future development of cognitive deterioration.