ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.     

A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase

The multilamellate glial sheath of mixed nerve roots of the sixth abdominal ganglion of crayfish contains numerous hemi-desmosomes which appear to attach glial lamellae to material in adjacent extracellular clefts. These junctions, which have been described in detail in an earlier report (Shivers and Brightman, '76), are irregular in shape, punctuate and may be as large as 1 mum in diameter. Surgical interruption of sixth ganglion nerve roots results in regeneration of motor axons and their multilamellate glial sheaths. As the glial processes grow and re-establish a highly organized axon sheath, hemi-desmosomes appear. These junctions are present at the advancing edge of glial processes as well as on their lateral margins. Developing hemi-desmosomes are characterized as a diffuse aggregation of 120-130 A intramembrane particles which are present three weeks following nerve section. As growth and reorganization of the sheath proceeds, the intramembrane particles appear to aggregate and form irregular clusters of varying dimensions. Regenerating nerves freeze-cleaved 8 to 16 weeks following surgery exhibit junctional particle aggregates similar to those in normal unoperated nerve roots. Origin of the intramembrane particles which comprise the junctional aggregated in unknown. Perhaps they are synthesized de novo by the regenerating glial cells or, they may be remnants of complexes which became dispersed following surgery. This is the first report of a freeze-fracture study of hemi-desmosome plasticity in an invertebrate nervous system.     

Infarct sizing with technetium-99m stannous pyrophosphate scintigraphy in dogs and man; relationship between scintigraphic and praecordial mapping estimates of infarct size in patients

The dynamics of changes in serum lipids (free fatty acids, free glycerol, triglycerides, total cholesterol, and phospholipids) were studied in male Wistar rats irradiated in an open experimental field with a daily dose of 15.48 mC.kg.--1 (60 R) up to a total exposure of 774.0 mC. kg.--1 (3,000 R). The resulting changes occurred in several periods. Initial period of 0--7 days included a drop in triglyceride level and a rise in free glycerol, total cholesterol, and phospholipids in both control and irradiated rats. The period of 14--25 days marked the appearance of serum hyperlipaemia. Between 25--50 days, the levels of the different fractions oscillated and existing changes became more pronounced. The general level of serum lipids during continuous gamma-irradiation exceeded that found in controls. Changes in control animals from experimental field reflected the influence of a changed environment. The modifying factor affecting both irradiated and control rats was night fasting prior to sacrificing the animals and, probably, also the presence of an infradian rhythm in some serum lipid fractions.     

Synovectomy and total joint arthroplasty for recurrent hemarthroses in the arthropathic joint in hemophilia

Severe hemophiliacs with intractable bleeding into one or more joints despite adequate clotting factor replacement therapy are difficult management problems. Synovectomy has controlled bleeding only in joints without significant arthritis destruction. Total joint replacements have been performed in arthropathic joints, but not when uncontrolled bleeding was a concurrent problem. This report describes a hemophiliac with uncontrolled bleeding into an arthritic knee who was successfully managed by combining synvectomy with total knee replacement.     

Electrocardiographic findings in patients undergoing amitriptyline treatment

Fifteen depressed patients were treated with amitriptyline in a dosage ranging from 75 mg to 200 mg/day for a minimum of three weeks, two of which were at a fixed dosage. Plasma samples were drawn 12 to 16 hours after the bedtime dose of medication for determination of amitriptyline and its metabolite nortriptyline. Electrocardiograms were taken prior to treatment and after three weeks of drug treatment. The only abnormality noted prior to treatment was bradycardia in one patient. After treatment, two patients exhibited nonspecific T-wave abnormalities. Otherwise, the electrocardiograms were unremarkable. A significant increase in heart rate (p less than 0.001) was noted. The mean increase in rate was sixteen beats per minute. Those patients having a rate change greater than sixteen beats per minute had significantly higher amitriptyline levels (p less than 0.05), and total tricyclic antidepressant levels (p less than 0.05), than those patients having a mean rate increase less than sixteen. While tricyclic antidepressants can produce multiple cardiac effects, the risk of cardiac morbidity and mortality is relatively low in patients undergoing tricyclic antidepressant treatment with moderate dosages.