Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide.

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.

Plantar fibromatosis of the heel in children: a report of 14 cases.

Based on a study of 14 patients younger than 16 years, we found that lumps on the plantar aspect of the anteromedial portion of the heel pad can safely be observed. Many remained small and asymptomatic, some disappeared, and two that required repeated excision biopsy did not subsequently recur. The six patients who were subjected to excision biopsy were found to have plantar fibromatosis. This very precise heel site is a characteristic situation for plantar fibromatosis in childhood.

A carbon monoxide irreducible form of cytochrome c oxidase and other unusual properties of the "monomeric" shark enzyme.

Contrary to previous reports, the functional and spectral properties of "monomeric" shark cytochrome c oxidases are not entirely similar to those of the "dimeric" beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a "g = 12" signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.

Biomechanical effects of gamma irradiation on fresh frozen allografts in vivo.

The effect of gamma irradiation on the mechanical properties of allograft in a rabbit model was examined. Tibial allografts were tested in torsion to failure to determine maximum torque and maximum stress. Statistically significant degradation in the torsional strength of irradiated tibias vs controls was demonstrated. Femoral allografts were orthotopically implanted into experimental rabbits and secured with an intramedullary pin. Rates of incorporation and failure patterns were significantly altered by irradiation. These data suggest that sterilization of fresh-frozen allografts with 2.5 Mrad or greater severely degrades the ability of the graft to resist torsion. The rate of incorporation of fresh-frozen allografts also may be dramatically decreased as a result of irradiation.

The site of the redox-linked proton pump in eukaryotic cytochrome c oxidases.

The electronic spectra of fully oxidized derivatives of some cytochrome c oxidase preparations are distinctly pH dependent. In general, the observed spectral shifts are greater in the case of pulsed derivatives compared to resting preparations and also, greater for preparations of the enzyme from shark skeletal muscle compared to beef heart. The low temperature near-infrared magnetic circular dichroism spectrum of the fully oxidized shark enzyme is not pH dependent in the experimental range, indicating the sensitivity of the visible region electronic spectrum to variation in pH to be due principally to changes at the heme a3-CuB chromophore. The results are discussed in relation to proposed mechanisms of proton translocation in cytochrome c oxidase.

Surgical correction of congenital kyphosis in myelomeningocele.

A progressive kyphosis in myelomeningocele patients may occur when the lesion is in the lower thoracic spine (i.e., a high-level lesion). Previously described techniques for this difficult problem have been fraught with problems of loss of fixation, recurrent deformity, implant failure, and wound breakdown. The technique described involves resection of the apical vertebra and proximal portion of the gibbus followed by fixation with a single U-shaped rod implanted distally into the lumbosacral spine. The rod is fixed to the thoracic spine using the Luque technique. No wires are used distally and the sacroiliac joints are not violated. This paper presents the first four patients who have undergone this form of spinal surgery. To date these patients have maintained a satisfactory result, and there has been no implant failure. Although this is a small series and with a short follow-up, the results support the use of this technique.

Is pinning in situ a safe procedure for slipped capital femoral epiphysis?

This report reviews the results of 37 patients with 46 involved hips treated with pinning for slipped capital femoral epiphysis. Particular attention was given to incidence of complications, including avascular necrosis, chondrolysis, and penetration of the joint. Of 46 hips with 1-year follow-up, there were no cases of chondrolysis. Of 23 hips with 2-year follow-up, there was only 1 case of avascular necrosis. Pin penetration, recognized at the time of surgery, produced no significant sequelae. Pinning can produce satisfactory results without a significant incidence of major complication.

Medial triceps flap coverage for an ulnar neuroma.

This paper reports on a technique for coverage of a posttraumatic lateral neuroma at the elbow. A 14-year-old male patient underwent exploration of the ulnar nerve 4 months after a laceration to the elbow. Following neurolysis and anterior submuscular transposition, the exposed neuroma was covered using a flap of medial triceps muscle. The flap required minimal mobilization, incurred little donor morbidity, and provided adequate coverage without the danger of creating a new area of compression.

Percutaneous screw fixation of intercondylar fracture of the distal humerus.

A greenstick intercondylar fracture of the distal humerus occurred in a 13-year-old boy. The articular surface of the trochlea was diastased nearly 5 mm. The fracture was reduced and fixed percutaneously using a 4.5-mm cannulated screw from a lateral approach.

Synergistic effects of vinblastine and recombinant interferon-beta on renal tumor cell lines.

Potential synergistic interactions between vinblastine (VBL) and recombinant interferon-beta (rIFN-beta) were assessed using median effect analysis. Calculation of the combination index demonstrated values less than 1 (indicating synergy) over a wide range of drug-induced growth inhibition for each of four different renal carcinoma cell lines (RCC). The degree of synergy observed could not be predicted from the morphology, doubling time, or relative sensitivity of the RCC lines to VBL and rIFN-beta. The optimal ratio of VBL to rIFN-beta in the combination appeared to be close to the ratio of the concentrations of each agent which yielded a 50% inhibition of growth. Although simultaneous presence of VBL and rIFN-beta in the culture medium was not required to demonstrate a synergistic effect, the minimum exposure time for rIFN-beta was determined to be 7 days. The uptake but not the egress of tritiated VBL into RCC cells was enhanced after growth for 4 days in 2.25 ng/ml of rIFN-beta. Median effect analysis can be shown to be independent of the mechanism of action of VBL and rIFN-beta and gives an indication of potential synergistic interactions over a wide range of drug effects. This method may prove useful in the selection of combinations of IFNs and antitumor drugs for clinical study.

Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus).

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2'-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.

Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.

Hemoglobin Deer Lodge (beta 2 His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Deer Lodge is an abnormal human hemoglobin with arginine substituted for histidine at the beta 2 position. X-ray crystallography of normal human hemoglobin has shown that the beta 2 residue is normally part of the binding site for 2,3-diphosphoglycerate. The substitution of arginine for histidine at beta 2 affects both the kinetics and equilibria of ligand binding. When stripped of anions, Hb Deer Lodge has an increased oxygen affinity and a decreased degree of cooperativity relative to Hb A. The alkaline Bohr effect is slightly increased and there are marked increases in oxygen affinity below pH 6 and above pH 8. In the presence of 2,3-diphosphoglycerate the cooperativity in increases to nromal and the pH dependence of oxygen binding is reduced. This contrasts with the enhanced Bohr effect seen for Hb A in the presence of organic phosphates. Due to enhanced anion binding at high pH, Hb Deer Lodge has a slightly lower oxygen affinity than Hb A at pH 9 in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate. Kinetic studies at neutral pH in the absence of organic phosphates revealed biphasicity in the rate of oxygen dissociation from Hb Deer Lodge, while approximately linear time courses were observed for Hb A. The fast phase of the oxygen dissociation kinetics shows great pH sensitivity, and organic phosphates increase the rate and percentage of the fast phase without greatly affecting the slow phase. The two phases are not resolvable at high pH. CO combination kinetics are much like those of Hb A except that "fast" and "slow" phases were apparent at wavelengths near the deoxy-CO isobestic point. We suggest that functional differences between the alpha and beta chains are enhanced in Hb Deer Lodge. After flash photolysis of the CO derivative, the percentage of quickly reacting material was slightly greater for Hb Deer Lodge than for Hb A. This may imply a somewhat greater tendency to dissociate into high affinity subunits. The substitution of arginine for histidine at beta 2 thus results in a macromolecule whose ligand-binding properties are significantly altered, the primary differences being expressed at high pH where Hb Deer Lodge binds anions more strongly than Hb A. The properties of Hb Deer Lodge are compared to those of other hemoglobin variants with substitutions at residues involved in binding of 2,3-diphosphoglycerate.