NADH Dehydrogenases: From Basic Science to Biomedicine

This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-1 is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membrane-associated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH–quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.     

Immunohistochemistry of Atrial Natriuretic Peptide in Brain Infarction

Atrial natriuretic peptide (ANP) was originally isolated from cardiac atria, and has potent natriuretic, diuretic, and vasorelaxant properties. It has been localized in neurons and astrocytes in the cerebral cortex and the white matter. We hypothesize that glial ANP may contribute to the regulation of cerebral blood flow in brain infarction. In order to elucidate this possible role, the immunohistochemistry of ANP was studied in cases of brain infarction and in other cases of brain trauma for comparison. A statistically significant increase in the number of ANP-immunoreactive glial cells (mainly astrocytes) was observed in the white matter surrounding the brain infarction compared with the intact area. No statistically significant increase in ANP-immunoreactive glial cell number was observed in the cerebral white matter from brain haemorrhage, contusion and control cases. Our results indicate that glial ANP may increase in number in brain infarction, and that it may be involved in the regulation of the cerebral blood flow in the infarcted area.     

A Randomized Controlled Trial of Multicomponent Exercise in Older Adults with Mild Cognitive Impairment

Background

To examine the effect of multicomponent exercise program on memory function in older adults with mild cognitive impairment (MCI), and identify biomarkers associated with improvement of cognitive functions.

Methodology/Principal Findings

Subjects were 100 older adults (mean age, 75 years) with MCI. The subjects were classified to an amnestic MCI group (n = 50) with neuroimaging measures, and other MCI group (n = 50) before the randomization. Subjects in each group were randomized to either a multicomponent exercise or an education control group using a ratio of 1∶1. The exercise group exercised for 90 min/d, 2 d/wk, 40 times for 6 months. The exercise program was conducted under multitask conditions to stimulate attention and memory. The control group attended two education classes. A repeated-measures ANOVA revealed that no group × time interactions on the cognitive tests and brain atrophy in MCI patients. A sub-analysis of amnestic MCI patients for group × time interactions revealed that the exercise group exhibited significantly better Mini-Mental State Examination (p = .04) and logical memory scores (p = .04), and reducing whole brain cortical atrophy (p<.05) compared to the control group. Low total cholesterol levels before the intervention were associated with an improvement of logical memory scores (p<.05), and a higher level of brain-derived neurotrophic factor was significantly related to improved ADAS-cog scores (p<.05).

Conclusions/Significance

The results suggested that an exercise intervention is beneficial for improving logical memory and maintaining general cognitive function and reducing whole brain cortical atrophy in older adults with amnestic MCI. Low total cholesterol and higher brain-derived neurotrophic factor may predict improvement of cognitive functions in older adults with MCI. Further studies are required to determine the positive effects of exercise on cognitive function in older adults with MCI.

Trial Registration

UMIN-CTR UMIN000003662 ctr.cgi&quest;function&hairsp;&equals;&hairsp;brows&amp;action&hairsp;&equals;&hairsp;brows&amp;type&hairsp;&equals;&hairsp;summary&amp;recptno&hairsp;&equals;&hairsp;R000004436&amp;language&hairsp;&equals;&hairsp;J.     

PGl2 analogue mitigates the progression rate of renal dysfunction improving renal blood flow without glomerular hyperfiltration in patients with chronic renal insufficiency.

Renal blood flow decreases with the progression of chronic glomerulonephritis (CGN). This disease induces medullary ischemia and further renal dysfunction in patients with chronic renal insufficiency (CRI). Prostacyclin (PGI2), with its vasodilative action, increases renal blood flow (RBF) without increasing glomerular filtration rate (GFR). We therefore examined the possibility that PGI2 would mitigate the progression of renal dysfunction by increasing RBF in patients with CRI. Sixteen patients with progressive renal insufficiency (serum creatinine: 2.14+/-0.89 mg/dl) due to CGN were prospectively chosen for this study. The blood pressure was already under control using calcium channel blockers before and during this study in nine hypertensive patients. In the first 6 months the patients received a low-protein (0.6 g/kg/day) and low-salt (5.0 g/day) diet. In the next 6 months they received 60 microg/day of PGI2 analogue (Beraprost sodium) orally. GFR was determined by 24-hour creatinine clearance, and effective renal plasma flow (ERPF) was determined by 99mTc-MAG3 scintigraphy. Glomerular capillary pressure, the resistance ratio of afferent and efferent arterioles (R(A)/R(E)), and the other hemodynamic parameters from Gomez's estimation equation were determined at the start of this study, just before the administration of Beraprost and at the end of the study. The levels of GFR and ERPF were 34.6+/-12.4 and 140.6+/-52.1 ml/min at the start of this study respectively, and decreased to 28.0+/- 12.0 and 115.6+/-45.3 ml/min after the first 6 months without Beraprost. The levels of GFR and ERPF stayed at 28.1+/-15.7 and 119.2+/-57.6 ml/min after the next 6 months with Beraprost in the same patients. R(A)/R(E) increased in the first 6 months from 7.9+/-3.6 to 10.8+/-8.6, but remained constant during 6 months of Beraprost administration, at 10.5+/-8.0. These data indicate that PGI2 analogue diminishes the vascular resistance of glomerular afferent and efferent arterioles regulating the decrease of renal blood flow without glomerular hyperfiltration, thus mitigating the progression rate of renal dysfunction.     

Analysis of Human Protein Replacement Stable Cell Lines Established using snoMEN-PR Vector

The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs. We have established and characterised two such partial protein replacement human cell lines (snoMEN-PR). Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as compared with conventional co-expression strategies. The snoMEN approach facilitates the study of mammalian proteins, particularly those that have so far been difficult to investigate by exogenous expression and has wide applications in basic and applied gene-expression research.     

Florigen is involved in axillary bud development at multiple stages in Arabidopsis

The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.     

NMR analysis of structure and function of snake neurotoxins

The 270-MHz proton-nmr spectra of short neurotoxins (erabutoxins from Laticauda semifasciata and cobrotoxin from Naja naja atra) and long neurotoxins (toxin B from Naja naja and α-bungarotoxin from Bungarus multicinctus) have been analyzed. The conformation of erabutoxin b in solution is largely consistent with the x-ray crystal analysis, although the environment of His-7 in solution is definitely different from that in the crystal. The pH-dependent transition has been found for toxin B, indicating that the conformation in neutral solution is different from that in the crystal as grown from acidic solution. The deuterium-exchange rates of the amide protons for the four neurotoxins have been measured. The order of structural rigidity is the same as the order of the irreversibility of neuromuscular block by neurotoxins.     

Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α₂βγδ (γ-AChR) or α₂βδ (-AChR). After birth, γ-AChRs are replaced by -AChRs (γ/-switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). -AChRs are suitable for strong adult muscle activities. Since the effect of the γ/-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included -subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development.