Autoantibodies to IL-1 alpha in sera from rapidly progressive idiopathic pulmonary fibrosis.

To clarify the clinical significance of autoantibodies to interleukin-1 alpha (IL-1 alpha autoantibodies) in rapidly progressive idiopathic pulmonary fibrosis (IPF), we measured the level of IL-1 alpha autoantibodies in serum of 11 patients on the first hospital day, when patients were admitted due to severe symptoms, and on the 21st hospital day. IL-1 alpha autoantibodies in serum were measured using radioimmunoassay, and the limitation of this assay for IL-1 alpha autoantibodies was 5 ng/ml. These antibodies were detected in 5 of 11 patients on the first hospital day. On the 21st hospital day, these antibodies were detected in all patients, and its level was increased compared with that on the first hospital day. IL-1 alpha autoantibodies that appeared in patients corresponded to that of IgG. The half life of exogenous autoantibodies was investigated following administration of autoantibody rich plasma obtained from healthy blood donors to 6 control patients (CP) and 6 progressive IPF patients. These autoantibody levels in their serum were less than 5 ng/ml before administration. Serum was obtained at the indicated time after administration of IL-1 alpha autoantibodies and the level of these autoantibodies in serum was measured, then the half life was calculated. Half life of exogenous IL-1 alpha autoantibodies in progressive IPF patients was significantly shorter than that in CP (71.3 +/- 31.8 hr vs 352.0 +/- 98.3 hr, p < 0.01). These findings suggested that IL-1 alpha autoantibodies were generated in response to the inflammatory process of rapidly progressive IPF and may act as a regulatory factor for IL-1 alpha.

A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats.

In view of a cytoprotective effect of elastase inhibitor on chemokine-mediated tissue injury, we examined the neuroprotective effect of ONO-5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine-induced neutrophil chemo-attractant (CINC)-1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC-1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO-5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC-1 protein and mRNA expression. ONO-5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC-1 contributed to inflammation in rat spinal cord injury and ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.

A non-binary direct digital synthesizer with an extended phase accumulator.

We describe a new direct digital synthesizer (DDS) in which output tuning resolution is flexibly controlled. The new DDS has an extended phase accumulator (EPA) controlled by two frequency control words; one determines the wave number within a single EPA operation cycle, and the other determines the length of the cycle. The EPA allows the DDS to provide jitter-free signals, the frequencies of which are given by arbitrary fractional expressions. (The denominator is fixed in conventional DDS that use normal phase accumulators.) Experimental results showed that the EPA worked well, allowing flexible output tuning resolution.

Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igkappa leader sequence.

IL-18 is a novel cytokine that induces interferon (IFN)-gamma secretion and plays an important role in antitumor immunity. In the present study, we constructed plasmid vectors encoding the murine mature IL-18 cDNA linked with the Igkappa leader sequence and the pro-IL-18 cDNA to estimate the efficacy of the mature IL- 18 vector and to evaluate IL-18--producing tumor cells as a tumor vaccine. Colon 26 cells were transfected with the abovementioned vectors or with vector alone (mock). Reverse transcription-polymerase chain reaction analysis showed increased expression of murine IL-18 cDNA in both mature IL-18 and pro-IL-18 transfectants in comparison to that in mock transfected cells. The ability of the culture supernatants of mature IL-18 transfectants to induce IFN-gamma secretion was extremely high (40-140 pg/10(6) cells) in comparison to that of pro-IL-18 transfectants (4-18 pg/10(6) cells). When injected into syngeneic BALB/c mice, the growth of mature IL-18 transfectants, but not pro-IL-18 transfectants, was significantly less than that in mock transfected cells ( P< .01, by ANOVA and analysis of covariance). In addition, injection of colon 26 or Meth-A cells into mice immunized with a mature IL-18 transfectant revealed acquired immunity. Depletion of natural killer cells did not affect the growth of transfectants. However, the growth inhibitory effects were partially abrogated following treatment with anti-CD4+ and anti-CD8+ antibodies. These data suggest that the rejection of mature IL-18/colon 26 cells was mediated through T-cell activation. Gene therapy using mature IL-18 transfectants containing a plasmid vector and the Igkappa leader sequence may be a useful tumor vaccine.

Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages.

BACKGROUND: Excessive lipid accumulation in macrophages plays an important role in the development of atherosclerosis. Recently, we discovered an adipocyte-specific plasma protein, adiponectin, that is decreased in patients with coronary artery disease. We previously demonstrated that adiponectin acts as a modulator for proinflammatory stimuli and inhibits monocyte adhesion to endothelial cells. The present study investigated the effects of adiponectin on lipid accumulation in human monocyte-derived macrophages. METHODS AND RESULTS: Human monocytes were differentiated into macrophages by incubation in human type AB serum for 7 days, and the effects of adiponectin were investigated at different time intervals. Treatment with physiological concentrations of adiponectin reduced intracellular cholesteryl ester content, as determined using the enzymatic, fluorometric method. The adiponectin-treated macrophages contained fewer lipid droplets stained by oil red O. Adiponectin suppressed the expression of the class A macrophage scavenger receptor (MSR) at both mRNA and protein levels by Northern and immunoblot analyses, respectively, without affecting the expression of CD36, which was quantified by flow cytometry. Adiponectin reduced the class A MSR promoter activity, as measured by luciferase reporter assay. Adiponectin treatment dose-dependently decreased class A MSR ligand binding and uptake activities. The mRNA level of lipoprotein lipase as a marker of macrophage differentiation was decreased by adiponectin treatment, but that of apolipoprotein E was not altered. Adiponectin was detected around macrophages in the human injured aorta by immunohistochemistry. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin suppressed macrophage-to-foam cell transformation, suggesting that adiponectin may act as a modulator for macrophage-to-foam cell transformation.

Rise in plasma leptin levels after stimulation of hypothalamic cholinoceptive neurons by neostigmine in rats.

To investigate the role of hypothalamic cholinergic neurons in the regulation of plasma leptin levels, we injected neostigmine, a cholinesterase inhibitor, or vehicle alone into the third cerebral ventricle in free moving male Wistar rats and then measured plasma leptin levels. The administration of neostigmine (5 x 10(-9) or 5 x 10(-8) mol) increased plasma leptin levels 3-6 h after stimulation in a dose-dependent manner, while intravenous injection of neostigmine (5 x 10(-8) mol) had no effect. Atropine (5 x 10(-8) mol) concomitantly injected with neostigmine (5 x 10(-8) mol) prevented neostigmine-induced increase in plasma leptin. The expression of leptin messenger ribonucleic acid (mRNA) in epididymal white adipose tissue was significantly increased at 4 and 6 h after neostigmine injection compared with that before the injection. Plasma levels of corticosterone were significantly increased at 30 min after stimulation with neostigmine and this increase was sustained for 6 h after stimulation. Furthermore, bilateral adrenalectomized rats showed no increase in plasma leptin levels after stimulation. In conclusion, stimulation of hypothalamic cholinoceptive neurons increased plasma leptin levels in rats by increasing leptin production in adipocytes. This increase may be due to an increase in glucocorticoids from the adrenal glands. These results suggest that plasma leptin levels can be regulated by hypothalamic cholinoceptive neurons.

Noninvolvement of hypertriglyceridemia and hyperleptinemia in blood pressure increases induced by dietary lard in rats.

We investigated whether hypertriglyceridemia and hyperleptinemia are involved in the development of increases in blood pressure induced by dietary lard. Rats received either chow alone or chow in which 50% of the energy content was from substituted lard. Each group was divided into two groups according to whether the diet included bezafibrate or not. In another series of experiments, rats were fed either chow alone or chow in which 50% of the energy content was from substituted lard, safflower oil, or sucrose. Systolic blood pressure (SBP) was measured every week during each 7-week feeding period. A steady-state plasma glucose method was used to determine insulin sensitivity after lard substitution with or without bezafibrate. After the 7-week feeding period, the plasma levels of glucose, immunoreactive insulin, triglyceride and leptin were measured. In rats fed with a high lard diet, SBP, plasma levels of immunoreactive insulin, triglyceride, leptin and steady-state plasma glucose concentrations significantly increased, compared with levels of these substances in controls. Bezafibrate treatment completely reversed these effects. In rats fed with a high safflower oil or a high sucrose diet, no significant change was seen in SBP and plasma immunoreactive insulin levels. However, the plasma triglyceride levels were increased by dietary lard or sucrose. Moreover, the plasma leptin level was also increased by dietary lard and safflower oil. Neither dietary hypertriglyceridemia nor hyperleptinemia were involved in the development of increases in blood pressure induced by dietary lard.

Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway.

BACKGROUND: Among the many adipocyte-derived endocrine factors, we found an adipocyte-derived plasma protein, adiponectin, that was decreased in obesity. We recently demonstrated that adiponectin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced expression of endothelial adhesion molecules and that plasma adiponectin level was reduced in patients with coronary artery disease (CIRCULATION: 1999;100:2473-2476). However, the intracellular signal by which adiponectin suppressed adhesion molecule expression was not elucidated. The present study investigated the mechanism of modulation for endothelial function by adiponectin. METHODS AND RESULTS: The interaction between adiponectin and human aortic endothelial cells (HAECs) was estimated by cell ELISA using biotinylated adiponectin. HAECs were preincubated for 18 hours with 50 microg/mL of adiponectin, then exposed to TNF-alpha (10 U/mL) or vehicle for the times indicated. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assays. TNF-alpha-inducible phosphorylation signals were detected by immunoblotting. Adiponectin specifically bound to HAECs in a saturable manner and inhibited TNF-alpha-induced mRNA expression of monocyte adhesion molecules without affecting the interaction between TNF-alpha and its receptors. Adiponectin suppressed TNF-alpha-induced IkappaB-alpha phosphorylation and subsequent NF-kappaB activation without affecting other TNF-alpha-mediated phosphorylation signals, including Jun N-terminal kinase, p38 kinase, and Akt kinase. This inhibitory effect of adiponectin is accompanied by cAMP accumulation and is blocked by either adenylate cyclase inhibitor or protein kinase A (PKA) inhibitor. CONCLUSIONS: These observations raise the possibility that adiponectin, which is naturally present in the blood stream, modulates the inflammatory response of endothelial cells through cross talk between cAMP-PKA and NF-kappaB signaling pathways.

Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients.

Adiponectin is a novel, adipose-specific protein abundantly present in the circulation, and it has antiatherogenic properties. We analyzed the plasma adiponectin concentrations in age- and body mass index (BMI)-matched nondiabetic and type 2 diabetic subjects with and without coronary artery disease (CAD). Plasma levels of adiponectin in the diabetic subjects without CAD were lower than those in nondiabetic subjects (6.6+/-0.4 versus 7.9+/-0.5 microg/mL in men, 7.6+/-0.7 versus 11.7+/-1.0 microg/mL in women; P<0.001). The plasma adiponectin concentrations of diabetic patients with CAD were lower than those of diabetic patients without CAD (4.0+/-0.4 versus 6.6+/-0.4 microg/mL, P<0.001 in men; 6.3+/-0.8 versus 7.6+/-0. 7 microg/mL in women). In contrast, plasma levels of leptin did not differ between diabetic patients with and without CAD. The presence of microangiopathy did not affect the plasma adiponectin levels in diabetic patients. Significant, univariate, inverse correlations were observed between adiponectin levels and fasting plasma insulin (r=-0.18, P<0.01) and glucose (r=-0.26, P<0.001) levels. In multivariate analysis, plasma insulin did not independently affect the plasma adiponectin levels. BMI, serum triglyceride concentration, and the presence of diabetes or CAD remained significantly related to plasma adiponectin concentrations. Weight reduction significantly elevated plasma adiponectin levels in the diabetic subjects as well as the nondiabetic subjects. These results suggest that the decreased plasma adiponectin concentrations in diabetes may be an indicator of macroangiopathy.

An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls.

Adipose tissue secretes a variety of proteins into the bloodstream. We have previously reported a novel cDNA, apM1 (adipose most abundant gene transcript 1), which is specifically and abundantly expressed in adipose tissue [1]. Primary structure analysis predicted that the apM1 gene product possesses significant homology to collagens VIII, X and complement factor C1q, and we named it adiponectin. In the current study, we analyzed characteristics of adiponectin in vitro and in vivo. Adiponectin protein was proved to be secreted into the medium when the cDNA was transfected to COS cells. Anti-adiponectin cross-reactivities were abundantly detected in the human plasma. In solid-phase binding assays, adiponectin specifically bound to collagen types I, III and V, which are present in vascular intima. Immunohistochemical analysis revealed that adiponectin was detected in the walls of the catheter-injured vessels but not in the intact vascular walls. These data suggest that adiponectin is a plasma protein produced by adipose tissue and accumulates in vascular walls when the endothelial barrier is injured.

Gender-related difference in relationship between insulin resistance and serum leptin level in Japanese type 2 diabetic and non-diabetic subjects.

It remains controversial whether or not a correlation exists between serum leptin levels and insulin resistance, and, if such a correlation does exist, whether it is independent of adiposity. To investigate the possible existence of an independent correlation, we have assessed serum leptin levels and insulin resistance in Japanese diabetic and non-diabetic subjects by means of Homeostatic Model Assessment (HOMA-R). Sixty-four Japanese patients with Type 2 diabetes mellitus (DM) (33 men and 31 women) and 53 sex-, age-, and body mass index (BMI)-matched non-diabetic adults (29 men and 24 women) were enrolled. The fasting plasma level of glucose (FPG) and the fasting serum levels of immunoreactive insulin (FIRI) and leptin were determined. Multiple linear regression analysis demonstrated that, in both male diabetic and male non-diabetic subjects, HOMA-R and BMI were independently correlated with serum leptin levels. In females, BMI, but not HOMA-R, was correlated to the serum levels of leptin in both groups. There was no statistically significant difference in the partial regression coefficients between male diabetic and male non-diabetic subjects. These results suggest that the correlation of HOMA-R to the serum levels of leptin in females is dependent on BMI. In males, the relationship between serum leptin levels and the insulin resistance was not affected by the extent of glucose intolerance.

Increased incidence of autoantibodies to interleukin-1a in rheumatoid arthritis with interstitial lung disease.

OBJECTIVE: To clarify the clinical significance of autoantibodies (auto-Ab) to interleukin-1alpha (IL-1alpha) in rheumatoid arthritis (RA) with interstitial lung disease (ILD), we examined the IL-1alpha auto-Ab level in serum of patients with RA with/without ILD. METHODOLOGY: We investigated the level of IL-1alpha auto-Ab in serum of 70 patients with RA with/without ILD and 40 control patients (CP). Levels of IL-1alpha auto-Ab were measured by radioimmunoassay, and serum was regarded as IL-1alpha auto-Ab positive at an auto-Ab level of more than 5 ng/mL. RESULTS: Interleukin-1alpha auto-Ab was detected in the serum of 30 out of 70 RA patients (42.9%), and six out of 40 CP (15%) (P < 0.05). Interleukin-1alpha auto-Ab were detected in the serum of 18 out of 32 patients with RA with ILD (56.2%) and 12 out of 38 patients with RA without ILD (31.5%). The positive rate of these autoantibodies in RA with ILD was significantly higher than that in RA without ILD (P < 0.05). Although C-reactive protein, immunoglobulin G, rheumatoid factor and rheumatoid arthritis particle agglutination levels in serum from patients with RA with ILD were not significantly different between the IL-1alpha auto-Ab-positive and -negative groups, the lactate dehydrogenase level (LDH) and AaDO, in the IL-1alpha auto-Ab-positive group were significantly higher than those in the negative group (LDH: P < 0.001, AaDO2: P < 0.05). CONCLUSION: These results suggest that IL-1alpha auto-Ab are generated in response to the immunoinflammatory process of ILD in RA, and these autoantibodies may neutralize and regulate the IL-1alpha activity.

Serum levels of soluble interferon Alfa/Beta receptor as an inhibitory factor of interferon in the patients with chronic hepatitis C.

Human serum contains a soluble form of interferon alfa/beta (sIFN alpha/beta) receptors, the functional and clinical significance of which has not been investigated in patients with chronic hepatitis C. In the present study, serum levels of sIFN alpha/beta receptor were assessed in 81 patients with chronic hepatitis C and correlated with the effectiveness of IFN therapy in these patients. Serum levels of sIFN alpha/beta receptor were significantly higher in patients with chronic hepatitis C than in healthy control patients (P <.0001). In these patients, serum levels of sIFN alpha/beta receptor were correlated with those of alanine transaminase (ALT) (P <.05), (2'-5')serum oligo(A) synthetase (2-5AS) (P <.0001), and pathological stages of liver fibrosis (P <.01). In 55 patients with chronic hepatitis C who underwent IFN therapy, there was an inverse correlation between the pretherapeutic serum levels of sIFN alpha/beta receptor and the rate of increase in serum levels of 2-5AS after the start of IFN (P <.01). Pretherapeutic serum levels of sIFN alpha/beta receptor were significantly lower in patients who showed sustained response to IFN therapy compared with those who did not respond to the therapy (P <.05). Multivariate analysis showed that low levels of serum sIFN alpha/beta receptor (

Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.

We isolated the human adipose-specific and most abundant gene transcript, apM1 (Maeda, K., et al., Biochem. Biophys. Res. Commun. 221, 286-289, 1996). The apM1 gene product was a kind of soluble matrix protein, which we named adiponectin. To quantitate the plasma adiponectin concentration, we have produced monoclonal and polyclonal antibodies for human adiponectin and developed an enzyme-linked immunosorbent assay (ELISA) system. Adiponectin was abundantly present in the plasma of healthy volunteers in the range from 1.9 to 17.0 mg/ml. Plasma concentrations of adiponectin in obese subjects were significantly lower than those in non-obese subjects, although adiponectin is secreted only from adipose tissue. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of adiponectin in humans.

Possible involvement of interleukin-1 in cyclooxygenase-2 induction after spinal cord injury in rats.

A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A2 (detected as thromboxane B2) and prostaglandin I2 (detected as 6-ketoprostaglandin F1alpha). Thromboxane B2 was predominant during the first 1 h, whereas the 6-ketoprostaglandin F1alpha level exceeded that of thromboxane B2 at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1alpha and -1beta detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1alpha into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F1alpha resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1alpha injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1alpha.

Up-regulation of vascular endothelial growth factor in response to glucose deprivation.

Vascular endothelial growth factor (VEGF), also known as a vascular permeability factor (VPF), is an endothelial specific mitogen and is a potent inducer of angiogenesis. Recently it has been reported that hypoxia induces VEGF mRNA expression in various cells. Since both oxygen and glucose are required for efficient production of energy, we examined the effect of glucose deprivation on VEGF mRNA expression and VEGF protein production in U-937 (a human monocytic cell line) cells. Both the mRNA expression and secretion of VEGF increased after exposure to low glucose. Addition of L-glucose, the L-stereoisomer of D-glucose, did not prevent the up-regulation of VEGF expression. The conditioned medium from glucose-deprived cells, followed by supplementation with glucose, did not up-regulate VEGF mRNA expression in U-937 cells. The low glucose-induced VEGF mRNA expression returned to the control level after supplementation with D-glucose. Furthermore, oligomycin, a mitochondrial ATP synthase inhibitor, increased VEGF protein production. The results suggest that the up-regulation of VEGF mRNA in U-937 cells in response to glucose deprivation is not mediated by autocrine factors from the cells nor is the osmotic change of the medium mediated by the deficiency of glucose metabolism in the cells. Our results also suggest that the intracellular ATP depletion due to glucose deprivation may be one of the causes for increased VEGF mRNA expression. We speculate that local hypoglycemia may act as an essential trigger for angiogenesis through the VEGF gene expression.

Age-related increase of autoantibodies to interleukin 1 alpha in healthy Japanese blood donors.

Although autoantibodies to interleukin-1 alpha (IL-1 alpha autoantibodies) are known to be present in sera of apparently healthy humans, their frequency of occurrence and significance are unclear. To determine the prevalence of detectable IL-1 alpha autoantibodies in normal human blood, we screened the plasma of blood donors (6290 subjects: 3977 men and 2313 women, ages 16 to 64 yr) by a radioimmununoassay which we developed using a method that could detect over 5 ng/ml. Moreover, we investigated immunoglobulin class of IL-1 alpha autoantibodies and also their function. IL-1 alpha autoantibodies were detected in 14.6% of the 6290 donors. Their frequency was higher in males than females (16.6% vs. 11.2%, p < 0.01) and increased with age in both sexes. The proportion of subjects with a high IL-1 alpha autoantibodies titers also increased with age. We showed that IL-1 alpha autoantibodies were of the IgG class and that they had neutralizing function to IL-1 alpha by receptor assay. Neutralizing activity was only shown in plasma with concentration of IL-1 alpha autoantibodies, the level of which was over 1000 ng/ml. The affinity of the IL-1 alpha autoantibodies in plasma was between 2.1 x 10(-10) and 1.2 x 10(-9) M (mean 6.4 x 10(-10)M). Our results provide a basis for comparison with IL-1 alpha autoantibodies prevalence between healthy states and disease states, and suggest that IL-1 alpha autoantibodies may play a significant role in modulating the effects of excessive IL-1 alpha at local site or in systemic regions.

Detection of immunoglobulin binding factor by enzyme-linked immunosorbent assay using two monoclonal antibodies.

Immunoglobulin binding factor (IgBF) found in human seminal plasma may be involved in suppressing antibody production against sperm in the female and male genital tracts. In the present study an enzyme-linked immunosorbent assay (ELISA) for IgBF was developed using monoclonal anti-IgBF antibodies. The sensitivity of the method was 20 pg/ml. The method was used to quantify IgBF in sera from women. In addition high levels of IgBF was found in cervical mucus of the uterus and in bronchial washings. The present results suggest that IgBF is found in tissues that are exposed to the external environment and may be a component of the local immunity system.

Stoichiometry of chargerin II (A6L) in the H(+)-ATP synthase of rat liver mitochondria.

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the A6L of mitochondrial DNA, may have a key role in the energy transduction by mitochondrial H(+)-ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the contents of chargerin II in the H(+)-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H(+)-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H(+)-ATP synthase.