Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.      

Cryosurgical ablation of accessory atrioventricular connections: a method for correction of the pre-excitation syndrome

Cryothermia, a new technique for definitive treatment of the pre-excitation syndrome, is described in two patients. The first patient presented with a normal P-R interval with a delta wave and reciprocating tachycardia. Preoperative electrophysiologic study suggested a free-wall atrioventricular connection on the left posterior atrioventricular (A-V) groove. At surgery, epicardial mapping confirmed the site of pre-excitation on the posterior left ventricular (LV) wall. An electrogram arising from the accessory pathway (AP) was recorded at the site of earliest ventricular activation. Interatrial delay combined with an apparently long accessory pathway to the ventricle caused the P-R interval to appear normal. Local pressure abolished pre-excitation. The site of early ventricular activation was cooled to -60 degrees C with a specially designed cryoprobe. All evidence of pre-excitation and arrhythmias disappeared. The second patient presented with a refractory reciprocating tachycardia and was found to have an AP in the septum capable of only retrograde conduction. Retrograde conduction was abolished by applying a temperature of 0 degrees C to the anulus at this site during tachycardia. Conduction over the AP and reciprocating tachycardia returned with rewarming. Ablation of the AP was obtained by applying a temperature of -60 degrees C for 90 seconds on two occasions to the same area. The His bundle was not injured.     

Effects of exercise on fluid exchange and body composition in man during 14-day bed rest

To determine the cause of the body weight loss during bed rest (BR), fluid balance and anthropometric measurements were taken from seven men (19-21 yr) during three 2-wk BR periods which were separated by 3-wk ambulatory recovery periods. Caloric intake was 3,073 +/- 155 (SD) kcal/day. During two of the three BR periods they performed supine isotonic exercise at 68% of VO2max on the ergometer for 1 h/day; or supine isometric exercise at 21% of maximal leg extension force for 1 min followed by a 1-min rest for 1 h/day. No prescribed exercise was given during the other BR period. During BR, body weight decreased slightly with no exercise (-0.43 kg, NS), but decreased significantly (P less than 0.05) by -0.91 kg with isometric and by -1.77 kg with isotonic exercise. About one-third of the weight reduction with isotonic exercise was due to fat loss (-0.69 kg) and, the remainder, to loss of lean body mass (-0.98 kg). It is concluded that the reduction in body weight during bed rest has two major components: First, a loss of lean body mass caused by assumption of the horizontal body position that is independent of the metabolic rate. Second, a loss of body fat content that is proportional to the metabolic rate.     

Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.     

Differences in tissue fatty acids and cholesterol of swine from different genetic backgrounds

A review of 1,000 consecutive coronary angiograms, most of them performed for evaluation of angina pectoris, yielded 9 examples of congenital anomalies of the coronary arteries. In 2 cases the angina may have been due to malposition of the left coronary artery or one of its branches. There were 2 cases of aberrant origin of the circumflex artery from the right coronary artery, 2 cases of aberrant left anterior descending artery, 3 cases in which all three major coronary branches arose from the right aortic sinus, and 2 cases of coronary artery fistulas. Malposition of the coronary artery should be considered as a possible cause of angina.