IL4Rα and ADAM33 as genetic markers in asthma exacerbations and type-2 inflammatory endotype.

BACKGROUND: Genetic markers of susceptibility to asthma exacerbations in adults remain unclear. OBJECTIVE: To identify genetic markers of asthma exacerbations, particularly in patients with type-2 inflammatory endotype. METHODS: In this observational study of patients enrolled in the Kinki Hokuriku Airway disease Conference multicenter study, frequency of exacerbations requiring systemic corticosteroids during 2 years after enrolment and associated risk factors was determined. For genetic marker analysis, interleukin-4 receptor α (IL4RA) rs8832 and a disintegrin and metalloprotease 33 (ADAM33) S_2 (rs528557), T_1 (rs2280091), T_2 (rs2280090), and V_4 (rs2787094) variants were included. Elevated serum periostin levels at enrolment (≥95 ng/mL, defined as type-2 inflammatory endotype) were considered in the analysis. RESULTS: Among 217 patients who were successfully followed up for 2 years after enrolment, 60 patients showed at least one asthma exacerbation during the 2 years. Airflow limitation (%FEV1 <80%) and recent exacerbations but not genetic variants were identified as risk markers of exacerbations. A total of 27 patients showed type-2 inflammatory endotype (serum periostin ≥95 ng/mL at enrolment) and subsequent exacerbations; risk factors in these patients were airflow limitation (odds ratio, 6.51; 95% confidence interval (CI): 2.37-18.6; P=.0003), GG genotype of IL4RA rs8832 (odds ratio, 4.01; 95% CI: 1.47-11.0; P=.007), and A allele of ADAM33 T_2 (odds ratio, 2.81; 95% CI: 1.05-7.67; P=.04) by multivariate analysis. In addition, GG genotype of IL4RA rs8832 was associated with type-2 endotype, whereas A allele of ADAM33 T_2 was associated with mixed type of eosinophilic/type-2 and neutrophilic inflammations. CONCLUSIONS AND CLINICAL RELEVANCE: IL4RA and ADAM33 variants may be risk markers of asthma exacerbations in type-2 inflammatory endotype. Precise endotyping may facilitate the identification of genetic risk markers of asthma exacerbations.

Visual methods to assess cold fingers and experimental verification.

Cold fingers is complaint of many people. To independently assess actual finger temperature, this paper uses prototype sensors to capture blood vessel width and blood flow rates. We verify their feasibility for future home healthcare use along with far infrared camera outputs. We elucidate the impact of three remedies, massage, hot cocoa, and shoulder exercises, on 7 subjects.

GLCCI1 variant accelerates pulmonary function decline in patients with asthma receiving inhaled corticosteroids.

BACKGROUND: In steroid-naive patients with asthma, several gene variants are associated with a short-term response to inhaled corticosteroid (ICS) treatment; this has mostly been observed in Caucasians. However, not many studies have been conducted for other ethnicities. Here, we aimed to determine the relationship between the annual decline in forced expiratory flow volume in one second (FEV1 ) and the variant of the glucocorticoid-induced transcript 1 gene (GLCCI1) in Japanese patients with asthma receiving long-term ICS treatment, taking into account the effect of high serum periostin levels, a known association factor of pulmonary function decline and a marker of refractory eosinophilic/Th2 inflammation. METHODS: In this study, 224 patients with asthma receiving ICS treatment for at least 4 years were enrolled. The effects of single-nucleotide polymorphisms (SNPs) in GLCCI1, stress-induced phosphoprotein 1 (STIP1), and T gene on the decline in FEV1 of 30 ml/year or greater were determined. RESULTS: Besides the known contributing factors, that is, the most intensive treatment step, ex-smoking, and high serum periostin levels (≥95 ng/ml), the GG genotype of GLCCI1 rs37973, and not other SNPs, was independently associated with a decline in FEV1 of 30 ml/year or greater. When patients were stratified according to their serum periostin levels, the GG genotype of rs37973 was significantly associated with blood eosinophilia (≥250/µl) in the high serum periostin group. CONCLUSIONS: A GLCCI1 variant is a risk factor of pulmonary function decline in Japanese patients with asthma receiving long-term ICS treatment. Thus, GLCCI1 may be associated with response to ICS across ethnicities.

The development and initial validation of a virtual dripping sweat rate and a clothing wetness ratio for use in predictive heat strain models.

This paper applies the heat balance equation (HBE) for clothed subjects as a linear function of mean skin temperature (t sk ) by a new sweating efficiency (η sw ) and an approximation for the thermoregulatory sweat rate. The equation predicting t sk in steady state conditions was derived as the solution of the HBE and used for a predictive heat strain scale. The heat loss from the wet clothing (WCL) area was identified with a new variable of 'virtual dripping sweat rate VDSR' (S wdr ). This is a subject's un-evaporated sweat rate in dry clothing from the regional sweat rate exceeding the maximum evaporative capacity, and adds the moisture to the clothing, reducing the intrinsic clothing insulation. The S wdr allowed a mass balance analysis of the wet clothing area identified as clothing wetness (w cl ). The w cl was derived by combining the HBE at the WCL surface from which the evaporation rate and skin heat loss from WCL region are given. Experimental results on eight young male subjects wearing typical summer clothing, T-shirt and trousers verified the model for predicting t sk with WCL thermal resistance (R cl,w ) identified as 25 % of dry clothing (R cl,d ).

Wearable blood flowmeter appcessory with low-power laser Doppler signal processing for daily-life healthcare monitoring.

A new appcessory for monitoring peripheral blood flow in daily life consists of a wearable laser Doppler sensor device and a cooperating smart phone application. Bluetooth Low Energy connects them wirelessly. The sensor device features ultralight weight of 15 g and an intermittent signal processing technique that reduces power consumption to only 7 mW at measurement intervals of 0.1 s. These features enable more than 24-h continuous monitoring of peripheral blood flow in daily life, which can provide valuable vital-sign information for healthcare services.

Prediction of mean skin temperature for use as a heat strain scale by introducing an equation for sweating efficiency.

The present paper made the heat balance equation (HBE) for nude or minimally clad subjects a linear function of mean skin temperature (t(sk)) by applying new equations for sweating efficiency (η(sw)) and thermoregulatory sweat rate (S(wR)). As the solution of the HBE, the equation predicting t(sk) was derived and used for a heat strain scale of subjects. The η(sw) was proportional to the reciprocal of S(w)/E(max) (S(w), sweat rate; E(max) maximum evaporative capacity) and the S(wR) was proportional to t(sk) with a parameter of the sweating capacity of the subject. The errors of predicted t(sk) from observations due to the approximation of η(sw) were examined based on experimental data conducted on eight young male subjects. The value of errors of t(sk) was -0.10 ± 0.42 °C (mean ± sample standard deviation (SSD)). We aim to apply the predicted t(sk) of a subject at a level of sweating capacity as a heat strain scale of a function of four environmental factors (dry- and wet-bulb temperatures, radiation, and air velocity) and three human factors (metabolic rate, sweating capacity, and clothing (≤0.2clo)).

Cytosolic phospholipase A2 alpha inhibitor, pyrroxyphene, displays anti-arthritic and anti-bone destructive action in a murine arthritis model.

OBJECTIVE: The aim of this study is to verify the crucial role of cytosolic phospholipase A2 alpha (cPLA2 alpha) in the pathogenesis of collagen-induced arthritis in mice and to determine the anti-arthritic effects of pyrroxyphene, a cPLA2 alpha inhibitor. METHODS: Pyrroxyphene was administered (p.o.) twice a day for 18 days at 30 and 100 mg/kg. Its effects on arthritic symptoms, bone destruction, cPLA2 alpha activity, levels of prostaglandin E(2) and leukotriene B(4), and mRNA expression of matrix metalloproteinase (MMP)-3, -8, -9, -13 and cyclooxygenase-2 (COX-2) were tested. RESULTS: cPLA2 alpha activity gradually increased and showed a correlation with the severity of arthritis. Pyrroxyphene strongly inhibited the incidence of arthritis and bone destruction. Moreover, it significantly inhibited both the increase in levels of cPLA2 alpha and eicosanoids as well as the mRNA expression of MMP-3, -8, -9, -13, and COX-2. CONCLUSION: These results demonstrate that cPLA2 alpha plays an important role in the pathogenesis of collagen-induced arthritis. Oral administration of pyrroxyphene achieved anti-arthritic activity through inhibition of cPLA2 alpha activity, which led to a reduction in eicosanoid levels and suppression of MMP and COX-2 mRNA expression. These results support a potential therapeutic role for cPLA2 alpha inhibitors in the treatment of human rheumatoid arthritis.

Cost of open versus laparoscopic appendectomy.

AIMS: There are several literatures on outcome variations between patients treated with an open appendectomy (OA) and a laparoscopic appendectomy (LA). However, there are no studies assessing differences in cost and outcome that adjust for age and hospital function or region. This study examines the differences in cost and procedure-related complications of OA and LA procedures. MATERIALS AND METHODS: This study contains 1703 appendectomy patients treated for appendicitis in 76 academic hospitals and 80 community hospitals. Demographic variables, clinical variables, length of stay (LOS), total charges (TC; US$) and complication rates were analyzed for both OA and LA procedures. The specific contributions of LA to LOS, TC, and complication rate were identified using multivariate analysis. RESULTS: 1469 (86.3%) patients underwent OA and 234 (13.7%) underwent LA. Complicated appendicitis was diagnosed in 13.1% of OA cases and 15.4% of LA cases. The complication rates were 3.4% in OA and 2.6% in LA (p=0.504). There were significant differences in LOS and TC by severity of appendicitis and by procedure type. After risk adjustment for the other study variables, LA was associated with a higher TC than OA ($1458, p0.001). However there were no significant differences in LOS or complication rates between the two treatment groups. CONCLUSIONS: This study suggests that LA increases cost, but has no significant impact on LOS or complication rates. However, other outcomes such as quality of life or subgroup analysis for obese patients are needed for a more complete economic analysis of OA and LA.

A begomovirus associated with Ageratum yellow vein disease in Indonesia: evidence for natural recombination between tomato leaf curl Java virus and Ageratum yellow vein virus-[Java].

A begomovirus (2747 nucleotides) and a satellite DNA beta component (1360 nucleotides) have been isolated from Ageratum conyzoides L. plants with yellow vein symptoms growing in Java, Indonesia. The begomovirus is most closely related to Tomato leaf curl Java virus (ToLCJV) (91 and 98% in the total nucleotide and coat protein amino acid sequences, respectively), although the products of ORFs C1 and C4 are more closely related to those of Ageratum yellow vein virus-[Java] (91 and 95% identity, respectively). For this reason, the begomovirus it is considered to be a strain of ToLCJV and is referred to as ToLCJV-Ageratum. The virus probably derives from a recombination event in which nucleotides 2389-2692 of ToLCJV have been replaced with the corresponding region of the AYVV-[Java] genome, which includes the 5' part of the intergenic region and the C1 and C4 ORFs. Infection of A. conyzoides with ToLCJV-Ageratum alone produced no symptoms, but co-infection with DNAbeta induced yellow vein symptoms. Symptoms induced in Nicotiana benthamiana by ToLCJV-Ageratum, ToLCJV and AYVV-[Java] are consistent with the exchange of pathogenicity determinant ORF C4 during recombination.

[Endobronchial hamartoma removed by bronchoscopic electrosurgical snaring].

A 61-year-old man was referred to our hospital, presenting with endobronchial mass in the right middle bronchus. Chest computed tomography (CT) showed a polypoid tumor. Bronchoscopy revealed a hard, smooth, whitish and pedunculated tumor obstructing the orifice of the both S4 and S5 segmental bronchi. We successfully removed this polypoid tumor via bronchoscopy using a biopsy forceps and electrosurgical snaring. The histological findings were compatible with chondromatous hamartoma. We recommend the endoscopic electrosurgical snaring to treat endobronchial hamartoma, especially when pedunculated, because this procedure is a minimal invasive technique.

Inhibition of antigen-induced arthritis in guinea pigs by a selective LTB4 receptor antagonist LY293111Na.

OBJECTIVE AND DESIGN: To investigate the role of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in the development and progression of antigen-induced arthritis (AIA) in guinea pigs and rats. METHODS: Arthritis was induced by injecting cationic amidated bovine serum albumin (aBSA) into the knee joint of immunized guinea pigs or rats. The effect of a potent and selective LTB4 receptor antagonist, LY29311INa (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy]benzoic acid sodium salt) was compared with those of indomethacin and dexamethasone. The effect of LY293111Na on adjuvant arthritis in rats was also examined. RESULTS: LY293 111Na (5 to 50 mg/kg b.i.d.) significantly inhibited knee joint swelling and histopathological changes of AIA in guinea pigs, but not in rats. Especially its protective effect against bone and cartilage destruction was substantial. In contrast, the cyclooxygenase inhibitor indomethacin significantly inhibited AIA in rats, but slightly inhibited in guinea pigs, while dexamethasone markedly inhibited AIA in both guinea pigs and rats. Increases of LTB4 and myeloperoxidase (MPO) activity were observed in the knee joint tissue of AIA guinea pigs, and LY293111Na dose-dependently inhibited the increase of MPO activity. Moreover, in adjuvant arthritic rats, LY293111Na showed slight inhibitory effect, while indomethacin showed marked inhibition. CONCLUSIONS: LTB4 but not PGE2 appeared to play important roles as an effective mediator in joint, particularly in cartilage and bone destruction of AIA in guinea pigs probably by inducing polymorphonuclear leukocytes (PMNs) chemotaxis to the joint tissue. In contrast, PGE2 but not LTB4 is an important mediator of arthritis in rats.

Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.

Although accumulating evidence indicates that cAMP response element-binding protein (CREB) phosphorylation mediates not only synaptic plasticity but also survival of certain neurons, it remains uncertain whether CREB phosphorylation induced after metabolic insult leads to CRE-mediated gene transcription and is involved in cell survival or not. In the present study, we clarified that (1) CREB phosphorylation and ischemic tolerance induced after preconditioning ischemia in the hippocampal neurons was abolished by MK801 administration in gerbil global ischemia model, (2) CREB phosphorylation induced after exposure to glutamate in cultured neurons was inhibited by removal of extracellular calcium, by MK801 and by an inhibitor of calcium-calmodulin-dependent protein kinase (CaMK) II and IV, (3) inhibitor of CaMK II-IV or CRE-decoy oligonucleotide suppressed upregulation of BCL-2 expression and accelerated neuronal damage after exposure to glutamate, and (4) CREB phosphorylation induced in the hippocampal neurons after ischemia and in cultured neurons after exposure to glutamate was followed by CRE-mediated gene transcription in transgenic mice with a CRE-LacZ reporter. Our results suggest that CREB phosphorylation in neurons after ischemia and exposure to glutamate is induced by NMDA receptor-gated calcium influx and subsequent activation of CaMK II-IV and that CREB phosphorylation after metabolic stress might show a neuroprotective response through CRE-mediated gene induction.

[A case of pulmonary inflammatory pseudotumor with hypergammaglobulinemia, elevated ANA, and uveitis].

A 63-year-old man presented with a chronic myeloproliferative disorder complicated with left pneumonia. His pneumonia was cured with antibiotics, but a nodular lesion remained in his chest radiographs together with hypergammaglobulinemia, a high titer of anti-nuclear antigen, and uveitis with secondary glaucoma. Specimens obtained by transbronchial lung biopsy showed a mixed accumulation of plasma cells, lymphocytes, and histiocytes as well as a spindle cell proliferation diagnosed as pulmonary inflammatory pseudotumor. The specimen did not show any recombination indicative of a heavy or a light chain of immunoglobulin in Southern blotting analysis. Oral prednisolone treatment improved the pulmonary nodular lesion, the abnormal laboratory data, and the uveitis. These findings suggest that much of the gammaglobulin produced by plasma cells in the inflammatory pseudotumor caused a variety of clinical symptoms.

Delayed, but marked, expression of apolipoprotein E is involved in tissue clearance after cerebral infarction.

Clearance of infarct tissue would be an important process for tissue repair after a stroke. Delayed clearance may hamper reconstitution of the blood-brain barrier and glial boundary formation. Recent growing evidence has indicated that apolipoprotein E (APOE), a major apoprotein, plays an important role in lipid transport and homeostasis in the brain. The tissue in the infarction contains abundant lipids must be removed for tissue clearance. In the current study, the authors investigated APOE expression after focal ischemia and the functional role of APOE in tissue clearance using APOE-knockout mice. Expression of APOE was delayed, but marked, in immunohistochemistry and immunoblotting 7 days after permanent focal ischemia. Macrophages were found to express APOE in the infarct center. Infarct size was similar after focal ischemia between wild-type and APOE-knockout mice, although there was no APOE protein expression in knockout mice. However, clearance of infarct tissue 2 weeks after ischemia was significantly delayed in APOE-knockout mice compared with wild-type mice. The current study supports current thinking that APOE is a key molecule for tissue remodeling in the brain. Clearance of damaged tissue may be one of the important functions of APOE in the brain.

Continuous surveillance of organochlorine compounds in human breast milk from 1972 to 1998 in Osaka, Japan.

The presence of chlorinated organic compounds in breast milk of lactating women living in Osaka Prefecture, Japan, has been followed since 1972. Following the highest concentration found at the start of the measurements, contamination by polychlorinated biphenyls (PCBs) and organochlorine pesticides, such as beta-HCH and DDT declined to about 3-13% of the peak levels by the mid-1970s and continued to decrease to low-level equilibrium states. This decline varies greatly with the chemical compound. In comparison with 1970s, when the contamination of breast milk was highest, the beta-HCH level fell to about 3%, DDT to about 7%, and PCBs to about 13%, representative of different half-lives in the environment and the particular persistence of PCB. In the case of chlordane, a termite insecticide, more than 10 years have passed since its use was prohibited in Japan, but it continues to be found in human breast milk, although at a low level.

Effects of leukotriene B4 receptor antagonist, LY293111Na, on antigen-induced bronchial hyperresponsiveness and leukocyte infiltration in sensitized guinea pigs.

OBJECTIVE AND DESIGN: LY29311 Na, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy] propoxy] -phenoxy]-benzoic acid sodium salt, is a novel leukotriene B4 (LTB4) receptor antagonist. Its effects on guinea pig models of asthma were compared with those of dexamethasone. METHODS: Effects of LY293111Na were tested in antigen (ovalbumin, OA)-induced bronchial hyperresponsiveness (BHR) and leukocyte accumulation in actively sensitized guinea pigs. Its effects on antigen-induced acute bronchoconstriction in passively sensitized guinea pigs were also studied. RESULTS: LY293111 Na (10 to 30 mg/kg p.o., 1 h before and 6 h after OA challenge) inhibited BHR to acetylcholine. LY293111 Na (3 mg/kg p. o.) significantly inhibited accumulation of neutrophils in bronchoalveolar lavage (BAL) fluid 24 h after antigen challenge but it did not inhibit accumulation of eosinophils and macrophages at any doses used. In contrast, dexamethasone (30 mg/kg p.o., 4 h before OA challenge) not only inhibited BHR but also reduced the infiltration of all three types of leukocytes. A significant increase of LTB4 levels in BAL fluid was noted at 3 and 15 min after the antigen challenge. LY293111Na did not inhibit antigen-induced acute bronchoconstriction in passively sensitized guinea pigs. CONCLUSION: These results indicate that LTB4 may participate in antigen-induced BHR but not in eosinophil infiltration and acute bronchoconstriction in guinea pigs.

Therapeutic time-window of a group IIA phospholipase A2 inhibitor in rabbit acute lung injury: correlation with lung surfactant protection.

OBJECTIVE: We attempted to determine whether group IIA secretory phospholipase A2 (sPLA2-IIA) blockade after the onset of lung injury exerted therapeutic efficacy in the treatment of oleic acid (OA)-induced acute lung injury by using S-5920/LY315920Na, a novel specific inhibitor of sPLA2-IIA, with special interest in the changes of lung surfactant. DESIGN: Prospective animal study. SETTING: University laboratory. SUBJECTS: Forty Japanese white rabbits. INTERVENTIONS: The rabbits, under anesthesia, were endotracheally intubated and mechanically ventilated and then were divided into the following groups: OA + vehicle groups, intravenous infusion of OA for the first 2 hrs (0.1 mL x kg(-1) x hr(-1)) with the addition of vehicle (1 or 2 hrs after OA administration, each n = 9, total 18 rabbits); OA + S-5920/LY315920Na groups, treated identically to the OA control with the addition of S-5920/LY315920Na (1 mg/kg bolus followed by infusion at 0.5 mg x kg(-1) x hr(-1)) after OA (1 or 2 hrs after OA administration, each n = 9, total 18 rabbits); saline control groups, treated with saline instead of OA with the addition of vehicle (1 hr after OA administration, 4 rabbits). Arterial blood gas, lung mechanics, lung inflammation, lung surfactant phospholipids, and production of inflammatory mediators in the lung were measured. MEASUREMENTS AND MAIN RESULTS: Treatment with S-5920/LY315920Na 1 hr after OA infusion, but not 2 hrs after infusion, significantly attenuated the lung injury, as estimated by hypoxemia, decreased lung compliance, pulmonary edema, and vascular permeability. The therapeutic efficacy was similar to that found in our previous pretreatment study. The treatment after 1 hr dramatically inhibited OA-induced surfactant degradation in the bronchoalveolar lavage fluid (BALF), without affecting the concentrations of thromboxane A2, leukotriene B4, and interleukin-8 in BALF. The degree of surfactant degradation in BALF paralleled well with the severity of the lung injury. Furthermore, recombinant human sPLA2-IIA reproduced the similar hydrolysis pattern of isolated surfactant in vitro, which was inhibited by S-5920/LY315920Na. CONCLUSIONS: Our results indicate that therapeutic blockade of sPLA2-IIA ameliorated lung dysfunction via protection of surfactant degradation in an animal model of acute lung injury, and they suggest a new strategy in treating clinical acute lung injury.

Effect of methylprednisolone on phospholipase A(2) activity and lung surfactant degradation in acute lung injury in rabbits.

Glucocorticoids are the most potent and widely used anti-inflammatory agents, but they are not particularly effective against early phase of acute respiratory distress syndrome. We investigated whether methylprednisolone, a synthetic glucocorticoid, could inhibit increase of phospholipase A(2) activity in the lung and lead to protection against a model of acute respiratory distress syndrome in rabbits. Infusion of oleic acid (0.1 ml/kg/h, i.v. for 2 h) provoked pulmonary hemorrhage and edema, protein leakage and massive neutrophil infiltration, resulted in severe hypoxemia and impaired lung compliance, accompanying the increase of phospholipase A(2) activity and interleukin-8, and degradation of surfactant in the bronchoalveolar lavage fluid. Infusion of methylprednisolone (60 mg/kg/h, i.v. for 30 min before the oleic acid and then 0.5 mg/kg/h, i.v. for 6 h) did not improve the above described lung injury induced by oleic acid, nor did it suppress phospholipase A(2) activity and degradation of surfactant in bronchoalveolar lavage fluid, while it strongly reduced interleukin-8 levels in both plasma and bronchoalveolar lavage fluid. We conclude that methylprednisolone did not attenuate oleic acid-induced acute lung injury and this can be explained partly by its failure to reduce the increase of phospholipase A(2) activity and the surfactant degradation in the lung, which might also account for its clinical ineffectiveness against early acute respiratory distress syndrome.

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I.

Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and alpha,beta-unsaturated gamma-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural gamma-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854-866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538-2546) that the gamma-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.