Bullous pemphigoid antigen. II. Isolation from the urine of a patient.

This report concerns the isolation of bullous pemphigoid antigen from the nondialyzable urinary components of a patient with the disease. The isolation was accomplished by ion exchange chromatography and gel filtration. Pemphigoid antigen was found to be a basic glycoprotein that on SDS gel electrophoresis showed two major bands, one in the 18,000 m. w. region and the second with a m. w. of 74,000. Between these two bands, two additional bands appeared; one of 35,000 daltons and the other of 68,000 daltons. The 18,000 m. w. band was eluted from the gel and rerun on SDS gels. These gels showed the 18,000 m.w. band and also the appearance of the 35,000 and 74,000 m.w. bands. This finding indicates that urinary pemphigoid antigen may exist both as a single monomeric form and in polymeric aggregates.     

(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.     

Lactose-proton symport by purified lac carrier protein

The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.     

A complex pattern of H2A phosphorylation in the mouse testis

Phosphorylation of H2A histones in mouse testis was examined using testis tubule cultures labeled with 32PO4. Histones were analyzed by two systems of two-dimensional polyacrylamide gel electrophoresis, followed by autoradiography of the gels. Of the 32PO4 detected in histones, 95% was incorporated by certain modified forms of the H2A variants H2A.1 and H2A.X. Phosphorylation sites were mapped to N- and C-terminal regions of the modified variants by SDS gel electrophoresis and autoradiography of peptides generated by cleavage of in vitro-labeled proteins with N-bromosuccinimide. Incorporation rates differed for N- and C-terminal regions from different modified forms, demonstrating a complex pattern of H2A phosphorylation in the mouse testis.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.     

Purification of the arom multienzyme aggregate from Euglena gracilis.

The arom multienzyme complex that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway has been purified up to 2000-fold from Euglena gracilis. The native arom aggregate has a molecular weight of approx. 249 000 based on a sedimentation coefficient of 9.5 and Stokes radius of 60 angstrom. A comparison between the arom aggregates of Neurospora crassa and Euglena gracilis and the possible phylogenetic relationships between the organisms are discussed.     

Response of fibrinolytic activity to venous occlusion.

Resting fibrinolytic activity and the response of the fibrinolytic system to venous occlusion were studied in a group of healthy middle-aged men. All subjects showed increased fibrinolytic activity but of varying degrees. There was a linear relationship between resting and occluded levels of fibrinolytic activity but many subjects with lower levels of fibrinolytic activity showed an anomalous response. Responses over the expected level were more common than unexpectedly low levels of response. Fibrinogen and plasminogen concentrations were inversely correlated with fibrinolytic activity.     

Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.