IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.     

A Comparative Study of the Structures of {alpha}-Glucans from Suspension-Cultured Nonglutinous and Glutinous Rice Cells

-Glucans (average mol wt, 1.3 ? 10⁴) extracted with perchloricacid from 8-day-old suspension-cultured nonglutinous (var. Sasanishiki)and glutinous rice (var. Miyakogane) cells were compared. Theresults of hydrolysis by alpha;-, ß- and iso-amylasesand methylation analysis of the -glucans suggested that theirbasic structures are almost the same. These -glucans are highly-branchedpolysaccharides with an average chain length of about 9–10,with exterior and interior chain lengths of about 6–7and 2–3, respectively. ¹Current address: Laboratory of Food Science, Faculty of Education,Hirosaki University, Hirosaki, Aomori 036, Japan. (Received April 27, 1987; Accepted March 2, 1988)     

Trimethylamine N-oxide respiration by aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Erythrobacter sp. OCh 114, an aerobic photosynthetic bacterium, had trimethylamine N-oxide (TMAO) reductase activity, which increased when the culture medium contained TMAO. The reductase was located in the periplasm. The bacteria grew anaerobically in the presence of TMAO. These results suggested that Erythrobacter OCh 114 has the ability to reduce TMAO through the respiratory chain. The TMAO respiration system of this organism was different from those of facultative purple photosynthetic bacteria in two respects: (a) TMAO reductase did not have activity to reduce dimethyl sulfoxide and (b) soluble c-type cytochrome, cytochrome c551, and cytochrome b-c1 complex appeared to be involved. The photochemical activity, which is usually inoperative in the anaerobic cell suspension, was restored by TMAO, suggesting that the photosynthesis and the TMAO respiration share a common electron transfer chain.     

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca²⁺ ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca²⁺. These findings suggest that even in the myosin-bound (open) state, Ca²⁺ may regulate actomyosin contractile properties via Tm.     

Improved Microbial Production of Colominic Acid,a Homopolymer of N-Acetylneuraminic Acid

A culture medium has been devised for producing colominic acid in improved yields. Major improvements were obtained by using sorbitol as a source of carbon, by adding phosphate in high concentrations, and by supplementing a limited amount of yeast extract. E. coli O 16: Kl: HNM produced approximately 3000 µg/ml of colominic acid on cultivation at 37°C for 46 hr with a liquid medium consisting of sorbitol (2.0%), (NH₄)₂SO₄ (0.5%), K₂HPO₄ (1.4%), MgSO₄·7H₂O (0.05%), and yeast extract (0.05%).

Isolation and purification by deproteinization with ammonium sulfate, precipitation with ethanol, and by column chromatography on anion exchange resins resulted in a pure colominic acid preparation devoid of internal ester linkages.

In producing colominic acid, strains forming S-type colonies were more active than those forming R-type colonies.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.