Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.     

Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis

High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates. Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template. The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus]. Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template. Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made. Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer). Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer. Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement.     

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.     

Guidelines for selective radiological assessment of inversion ankle injuries.

A prospective study was performed to establish definitive guidelines for selective use of radiography in the assessment of inversion ankle injuries. Five hundred patients were included, representing 3.2% of the workload of the department during the study period. There were 379 soft tissue injuries, 56 malleolar fractures, 40 avulsion fractures, 21 fractures at the base of the fifth metatarsal, and four calcaneal fractures. Multiple logistic regression identified distal fibular tenderness, age, and ability to bear weight as the most important clinical variables in predicting important fractures (p less than 0.001). A policy of requesting x ray examination of only those patients with distal fibular tenderness or inability to bear weight or aged over 60, with a further proviso that no foot radiographs should be obtained, would produce a 60% reduction in ankle radiography in this centre without detriment to patient care.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Mechanisms of blood flow during pneumatic vest cardiopulmonary resuscitation

Mechanisms of blood flow during cardiopulmonary resuscitation (CPR) were studied in a canine model with implanted mitral and aortic flow probes and by use of cineangiography. Intrathoracic pressure (ITP) fluctuations were induced by a circumferential pneumatic vest, with and without simultaneous ventilation, and by use of positive-pressure ventilation alone. Vascular volume and compression rate were altered with each CPR mode. Antegrade mitral flow was interpreted as left ventricular (LV) inflow, and antegrade aortic flow was interpreted as LV outflow. The pneumatic vest was expected to elevate ITP uniformly and thus produce simultaneous LV inflow and LV outflow throughout compression. This pattern, the passive conduit of "thoracic pump" physiology, was unequivocally demonstrated only during ITP elevation with positive-pressure ventilation alone at slow rates. During vest CPR, LV outflow started promptly with the onset of compression, whereas LV inflow was delayed. At compression rates of 50 times/min and normal vascular filling pressures, the delay was sufficiently long that all LV filling occurred with release of compression. This is the pattern that would be expected with direct LV compression or "cardiac pump" physiology. During the early part of the compression phase, catheter tip transducer LV and left atrial pressure measurements demonstrated gradients necessitating mitral valve closure, while cineangiography showed dye droplets moving from the large pulmonary veins retrograde to the small pulmonary veins. When the compression rate was reduced and/or when intravascular pressures were raised with volume infusion, LV inflow was observed at some point during the compressive phase. Thus, under these conditions, features of both thoracic pump and cardiac pump physiology occurred within the same compression. Our findings are not explained by the conventional conceptions of either thoracic pump or cardiac compression CPR mechanisms alone.     

Lymphocyte entry into inflammatory tissues in vivo. Qualitative differences of high endothelial venule-like vessels in sponge matrix allografts vs isografts

Sponge matrix allografts and isografts become extensively encapsulated and neovascularized after s.c. implantation. Sponge allografts acquire alloantigen-reactive T lymphocytes, whereas sponge isografts fail to do so, even though these T cells are continuously circulating in the peripheral blood. We have investigated the possibility that the vascular endothelia regulates lymphocytic accumulation in sponge matrix implants. In normal lymph nodes, specialized high endothelial venules (HEV) regulate lymphocyte extravasation from the blood. We have now identified HEV-like vessels in sponge matrix allografts. These vessels are operationally defined as "HEV-like" in that they react with mAb MECA 325 which identifies murine HEV, and bind lymphocytes in ex vivo adhesion assays. In contrast, sponge isografts contain MECA 325 reactive vessels that are significantly smaller than those found in allografts. Further, vessels of sponge isografts do not readily bind lymphocytes in ex vivo adhesion assays. Immunohistologic analysis also revealed that the small MECA 325+ vessels present in sponge isografts are consistently found in close proximity to nerve bundles. Although this MECA 325 reactive vessel-nerve bundle association is also observed in sponge allografts, large MECA 325 reactive vessels are widely distributed in allografts. Our data suggest that small, poorly adhesive MECA 325 reactive vessels develop in sponge isografts and allografts, possibly under the influence of local nerve tissue. These vessels respond to regional alloimmune responses by developing into the larger HEV-like vessels capable of binding lymphocytes in sponge allografts. The value of this experimental system as an in vivo model to evaluate mechanisms involved in neovascularization and endothelial differentiation is discussed.