Histochemical study of mitochondrial enzymes in cerebellar cortex of macular mutant mouse,a model of Menkes kinky hair disease

The cerebellar Purkinje cells in the hemizygote of the macular mutant mouse contain numerous abnormal mitochondria which show a marked decrease in cytochrome c oxidase activity. Using histochemical methods we studied the activity of other mitochondrial enzymes, such as NADH diaphorase and succinic dehydrogenase, in the cerebellar cortex of this mutant mouse. Such activities were markedly increased in the Purkinje cells, especially in the soma and stem dendrite, from 10 days after birth in the hemizygote as compared with findings in normal littermates. These results were considered to be due to an increased number of abnormal mitochondria.Supported by the Research Grants nos. 2A-5-03 and 3B-1-04 for Nervous and Mental Disorders from the Ministry of Health and Welfare, and by Grant no. 04670492 of the Ministry of Education of Japanese Government     

Monitoring myocardial reperfusion injury with NADH fluorometry.

Using NADH fluorometry to monitor myocardial metabolism, the mechanism of reperfusion injury was investigated after the delivery of an experimental reperfusate. Using an isolated working heart preparation, rat hearts underwent 15 min of global ischemia at 37 degrees C. Following the ischemic insult, an oxygenated enriched reperfusion solution was given for 5 min. The hearts were then returned to a working state and aortic flow recorded to evaluate recovery. NADH levels were monitored throughout the experiment with a fluorometer and glycogen, AMP, ADP, and ATP were measured biochemically pre- and postischemia, after reperfusion and after recovery. In this study, reperfusion injury was best abated by an enriched reperfusate. Our results indicate the mechanism for this amelioration is not high-energy phosphate replenishment. Rather, as indicated by NADH fluorescence, the hearts attain an intermediate level of metabolism that permits glycogen to be restored and functional recovery to be improved.     

Peculiarities of external respiration as a marker of activity of cell respiratory enzymes in rat brain

Rats with catatonic freezing are characterized by low motor and explorative activity in the open field test and specific pattern of external respiration, so-called strenuous respiration, which is accompanied by a higher activity of the respiratory enzymes succinate dehydrogenase and NADH dehydrogenase in the hippocampus and motor cortex. No dependencies on the brain structure and animal age are noted. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 138–140, August, 1997     

Deleterious effect of exogenous angiotensin-I on the extent of regional ischaemia and its inhibition by captopril

The endogenous activity of the local renin-angiotensin system(RAS) and the anti-ischaemic properties of captopril were investigatedin electrically driven rabbit Langendorff hearts (constant pressure:70 cmH₂O, Tyrode solution, Ca²⁺ 1.8 mmol.l^–1). Cumulativeconcentration-response curves showed no significant difference(P>0.05) between the reduction of the global coronary flow(CF) by exogenous angiotensin-I or angiotensin-II (EC₅₀ = 10^–10mol.l^–1). It is concluded that the local RAS in isolatedrabbit hearts is highly sensitive, whereas its endogenous activityis very low due to very low endogenous angiotensin-I content.Myocardial ischaemia (MI) was induced by the occlusion of aleft coronary artery branch and MI was quantified from NADHsurface fluorescence photography. MI was significantly enlarged(+35%) (P <0.05) by exogenous angiotensin-I (6x10^–9mol.l^–1). The reduction in CF and the increment in MIby angiotensin-I could be completely prevented by adding captoprilat a low concentration (10^–6 mol.l^–1) to the perfusionbuffer. In the absence of exogenous angiotensin-I, captoprilalone (10^–6 mol.l^–1) neither significantly enhancedCF (P >0.05), nor diminished MI (P >0.05), supportingthe finding of very low endogenous activity of tile local RASin this model. We, moreover, conclude that at a low concentration(10^–6 mol.l^–1) captopril does not possess directcardioprotective properties independent of its ACE inhibitingaction.     

The 49 K subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria: primary structure of the gene and the protein

Summary The primary structure of the 49 K subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The sequence lengths correlate to a molecular mass of 54002 daltons for the preprotein and 49239 daltons for the mature protein. The presequence consists of 42 amino acids of typical composition for sequences which target nuclear-encoded proteins into mitochondria. The mature protein consists of 436 amino acids and shows 64% similarity to a 49 K subunit of bovine heart NADH:ubiquinone reductase and 33% to a predicted translation product of an open reading frame in the chloroplast DNAs of Marchantia polymorpha and Nicotiana tabacum. Evidence for an iron-sulfur cluster in the subunit is discussed.     

缺氧性肺动脉高压时一氧化氮合酶、左旋精氨酸及亚硝基左旋精氨酸甲酯的研究

目的研究一氧化氮（ＮＯ）在缺氧性肺动脉高压（ＨＰＨ）发病中的作用。方法用烟酰胺腺嘌呤二核苷酸磷酸—黄递酶（ＮｉｃｏｔｉｎａｍｉｄｅＡｄｅｎｉｎｅＤｉｎｕｃｌｅｏｔｉｄｅＰｈｏｓｐｈａｔｅＤｉａｐｈｏｒａｓｅ，ＮＡＤＰＨｄ）和免疫组化ＡＢＣ方法检测原生型和诱生型一氧化氮合酶（ｃＮＯＳ、ｉＮＯＳ）在正常和缺氧大鼠肺内的表达和分布，同时观察左旋精氨酸（Ｌａｒｇ）和亚硝基左旋精氨甲酯（ＬＮＡＭＥ）对正常和缺氧大鼠肺循环的影响。结果ＨＰＨ组，ＮＡＤＰＨｄ阳性反应见于大血管内皮、平滑肌、支气管粘膜上皮及小血管内皮和平滑肌中，而后者在正常时未见阳性反应；ｃＮＯＳ在肺血管内皮和支气管粘膜上皮中的表达明显减弱，甚至消失，而正常时不表达ｉＮＯＳ的肺血管内皮和血管、支气管平滑肌在ＨＰＨ组出现了阳性表达；缺氧时补充Ｌａｒｇ和ＬＮＡＭＥ，与单纯缺氧相比，对右心室肥大和肺血管重建无影响。结论缺氧时ｃＮＯＳ被抑制，可能对ＨＰＨ的形成具有一定的作用；而ｉＮＯＳ的诱导表达，则可能对ＨＰＨ的形成具有阻止作用。     

Purification of the NADH reductase component of the steroid 9α-hydroxylase fromMycobacterium fortuitum

The NADH reductase component of the steroid 9α-hydroxylase fromMycobacterium fortuitum was purified to homogeneity. Recovery of the enzyme from the 50≈60% ammonium sulfate saturated fraction was 49%, with a purification factor of 100-fold. The NADH reductase has a relative molecular mass of 60 KDa as determined by SDS-PAGE. The absorption maxima at 410 and 450 nm indicate the presence of iron-sulfur group and flavin. These prosthetic groups seemed to function as redox groups that transfer electrons from NADH to the following protein. The K_M value for NADH as substrate was 68 μM. The NH₂-terminal amino acid sequence of the reductase was determined as Met-Asp-Ala-Ile-Thr-Asn-Val-Pro-Leu-Pro-Ala-Asn-Glu-Pro-Val-His-Asp-Tyr-Ala-Thr. This sequence does not show a homology with the NH₂-terminal sequences reported for the reductase component of other monooxygenases, suggesting that the NADH reductase component of the steroid 9α-hydroxylase system is novel.     

Correlated, simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation

Current forms of brain monitoring, such as electroencephalography (EEG), have had limited clinical utility. The EEG records spontaneous cerebrocortical activity and thus is an indirect indicator of metabolic demand and, to a lesser extent, an indicator of mismatch of supply versus demand. Ischemia modulates EEG activity in ways that can usually be detected, but EEG patterns can be similarly modulated by many other factors, including temperature and pharmacologic manipulation. This in vivo study in physiologically monitored animals evaluated the use of correlated optical spectroscopy, performed with an instrument having a fiberoptic light-guide bundle in contact with the cerebral cortex, for the simultaneous monitoring of cerebrovascular oxygen availability and intracellular oxygen delivery. A highly specific monitor of cerebral intracellular oxygen supply, the cerebrocortical intramitochondrial NADH redox state, was monitored in vivo with a fluorescence technique. Absorption spectroscopy was used concurrently to monitor hemoglobin content (blood volume) and oxygen saturation in the microcirculation. Correlated changes in optical signals from cerebrocortical NADH and hemoglobin were studied in a swine model (n=7) of nitrogen hypoxia. Measurements were made at four wavelengths with a time-division, multiplexed fluorometer/reflectometer. Because the NADH fluorescence signal at 450 nm is affected by local changes in blood volume, a corrected fluorescence signal is usually calculated. In previous studies, where only two wave lengths have been measured, attempts at correction were based on reflectance at the excitation wavelength (366 nm). We compared estimators of changes in microcirculatory blood volume using reflection at two wavelengths: 366 nm and 585 nm, the wavelengths for maximum and isobestic absorption. The results of the studies were as follows: (1) during transient hypoxia, NADH and local hemoglobin saturation signals changed in concert with arterial pulse oximetry, with changes in NADH lagging behind changes in saturation by an average of 5.3 seconds; (2) after hypocapnic ventilation to a mean Paco ₂ of 20.2 ± 0.8 mm Hg, NADH increased by 11.5 ± 8.7% (as compared with maximal change during anoxia), local hemoglobin saturation decreased by 7.7 ± 6.4%, and local blood volume decreased by 12.5 ± 13%, while arterial SpO₂ was unchanged; (3) our two measures of local blood volume were closely correlated during carbon dioxide perturbations, but poorly correlated during hypoxic perturbation; and (4) NADH fluorescence provided a more rapid, sensitive indicator of oxygen deprivation than did the EEG. During transient hypoxia, EEG changes occurred 57.4 ± 10.4 seconds after the onset of decline in local hemoglobin saturation, after NADH had completed 50% of its maximal increase.This work was supported in part by research grants from the NIH (GM34767), the Academic Senate of the University of California, and the UCSF Anesthesia Research Foundation.     

Ion Transport and Energy Metabolism in Brain

Abstract

The kinetics of extracellular K⁺ activity was compared to the availability of energy in the cortex of rats and gerbils exposed to anoxia, hypoxia, spreading depression, and ischemia.

A combined K⁺/DC surface electrode was used alone or together with a fiber optic light guide in various experiments. All experiments were done on slightly anesthetized animals that were practically awake. The results can be summarized in the following conclusions: (1) Under conditions such as hypoxia or ischemia, K⁺₀ showed a two-phase efflux kinetics, and a transition or critical point was reached where the response proceeds at a higher rate. This critical point was in the range of 10-16 mEq/1 potassium. (2) Availability of oxygen is necessary but not sufficient for a full rate of recovery from a long-term oxygen-deprivation insult. (3) There is an energy debt or energy-transduction bottleneck fonned during a prolonged O₂ insufficiency. This debt is reversed slowly during the recovery phase even when full restitution of O₂ supply and blood flow has occurred.     

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca²⁺]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca²⁺], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca²⁺] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca²⁺], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca²⁺] and TMRE for Ψ_m or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca²⁺] concentration was 1.03 µM. This 1 µM cytosolic Ca²⁺ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca²⁺] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca²⁺. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.