Isolation and characterization of folded fragments released by Staphylococcal aureus proteinase from the non-histone chromosomal protein HMG-1

HMG-1 was isolated from newborn calf thymus without exposure to overt denaturing conditions. The purified protein was digested under several solvent conditions with the proteinase (endoproteinase GluC) from Staphylococcus aureus strain V8. We found that the preferred site of attack by the enzyme on HMG-1 was influenced markedly by ionic strength and temperature. In 0.35 M NaCl/50 mM Tris-phosphate (pH 7.8) at 37 degrees C, cleavage near the junction between the A and B domains is predominant, as previously reported by Carballo et al. (EMBO J. 2 (1983) 1759-1764). However, in 50 mM Tris-phosphate (pH 7.8) lacking NaCl and at 0 degrees C, cleavage between the B and C domains strongly predominates. Three major products of the digestions were purified and characterized. The fragment consisting of domains B and C was found by circular dichroism to contain a substantial amount of helix. This re-emphasizes the importance of avoiding overt denaturing conditions when working with members of the HMG-1 family.     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.     

The effect of ACTH on the blood clearance rate of aldosterone, cortisol, 17 alpha-hydroxyprogesterone and 17 alpha, 20 alpha-dihydroxy-4-pregnen-3-one in the sheep

The blood clearance rate (BCR) of aldosterone, cortisol, 17 alpha-hydroxyprogesterone (17 alpha OHP) and 17 alpha, 20 alpha-dihydroxy-4-pregnen-3-one (17 alpha 20 alpha OHP) has been measured in conscious sheep prior to and after 5 or 6 days ACTH treatment. ACTH increased the BCR of cortisol but did not change the BCR of the other three steroids. 17 alpha OHP had a BCR greater than liver blood flow suggesting extra-hepatic metabolism. In vivo conversion of 17 alpha OHP to 17 alpha 20 alpha OHP by ovine red cells has been shown to be a significant site of this metabolism. It is suggested that this conversion of 17 alpha OHP to 17 alpha 20 alpha OHP may be important in the expression of the "hypertensionogenic" effect of 17 alpha OHP.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

Thievery,home ranges,and nestmate recognition inEctatomma ruidum

Summary Thievery of food items among colonies of a ponerine ant,Ectatomma ruidum was common; nonnestmates in colonies or near the colony entrances receive incoming food items and carry them to their own colony. In our study area 7 of 10 colonies were victimized by thief ants. Colonies have discrete home ranges and home range size is correlated with the number of workers in the colony. Worker ants discriminate nestmates from non-nestmates when non-nestmates are presented at colony entrances, but individuals from different colonies were not observed to engage in agonistic interactions away from nest entrances. Non-nestmates gain entrance to colonies when the entrance is unguarded. Many instances of non-nestmates being removed from colonies by residents were observed. The costs and benefits of theft under these circumstances are considered.