Antebrachiometacarpal arthrodesis in five dogs.

CASE REPORTS: Five dogs (4 with severe carpal contracture, 1 with a chronically infected carpal joint) underwent antebrachiometacarpal arthrodesis. Excision of all carpal bones, except the accessory carpal bone, was done, either because of persistent infection or to allow the manus to be arthrodesed in a functional position. All five dogs developed varying degrees of soft tissue swelling of the surgical site following surgery. All five arthrodeses achieved complete osseous union within 4-67 weeks. The immediate postoperative distal radiometacarpal frontal plane angulation ranged from 1° to 19° (mean ± SD: 7 ± 8°). The immediate postoperative distal radiometacarpal sagittal plane angulation ranged from 6° to 26° (mean ± SD: 17 ± 9°). Plate coverage of the secured metacarpal bone(s) ranged from 75% to 87% (mean ± SD: 80 ± 4%). Infection necessitated plate removal in four dogs, 3-17 (mean ± SD: 8 ± 6) months following surgery and 0-15 (mean ± SD: 5 ± 7) weeks following radiographic documentation of complete osseous union. CONCLUSION: Despite one dog having marked elbow incongruency and degenerative joint disease and one dog having an ipsilateral radial nerve deficit, all five dogs improved and had acceptable limb function at the time of the final evaluation, which ranged from 25 to 296 (mean ± SD: 99 ± 111) weeks following surgery.

HLA Class II Antibody Activation of Endothelial Cells Promotes Th17 and Disrupts Regulatory T Lymphocyte Expansion.

Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab')2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3(bright) regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells.

Thinning, movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis.

Plasticity of residual cortical tissue has been identified as an important mediator of functional post-stroke recovery. Many studies have been directed toward describing biochemical, electrophysiological, and cytoarchitectural changes in residual cortex and correlating them with functional changes. Additionally, after neonatal stroke the thickness of residual tissue can change, the tissue can move, and tissue can fill in the stroke core. The purpose of the present study was to systematically investigate and document possible gross morphological changes in peri-infarct tissue after forelimb motor cortex stroke in the adult rat. Rats received a unilateral forelimb motor cortex stroke of equivalent size by pial strip devascularization or photothrombotic occlusion and were then examined using histology or magnetic resonance imaging (MRI) at 1 h, 1, 3, 7, 14, or 31 days post-stroke. Middle cerebral artery occlusion was used as a control stroke procedure. Decreases in cortical thickness, volume, and neural density were found to extend far beyond the stroke infarct and included most of the sensorimotor regions of the stroke and intact hemispheres. Movement of residual tissue towards the infarct was observed and confirmed using anatomical markers placed in intact cortical tissue at the time of stroke induction. The results are discussed in relation to the idea that extensive time-dependent morphological changes that occur in residual tissue must be considered when evaluating plasticity-related cortical changes associated with post-stroke recovery of function.

Examination of synovial fluid and serum following intravenous injections of hyaluronan for the treatment of osteoarthritis in dogs.

A randomized, blinded, prospective clinical trial was performed to determine the effects of intravenous (i.v.) administration of hyaluronan sodium (HA) on serum glycosaminoglycans (GAG) concentrations, synovial fluid (SF) hyaluronan concentrations and viscosity in dogs treated for unilateral rupture of the cranial cruciate ligament. Twenty-two dogs undergoing tibial plateau leveling osteotomy were used in this study. Synovial fluid from both stifles and serum were collected prior to surgery and at 2, 4, and 8 weeks following surgery. Dogs received either 1.0 ml (10 mg) of sodium hyaluronate (treatment group 1; n = 10) or equal volume of 0.9% NaCl (treatment group 2; n = 12), i.v. immediately, 2 and 4 weeks following surgery. Synovial fluid viscosity was evaluated using a magnetically driven, acoustically tracked, translating-ball rheometer. Synovial fluid HA disaccharide content was measured by fluorophore-assisted carbohydrate electrophoresis. Serum GAG concentrations were measured by alcian blue spectrophotometric assay. Data were analyzed using a Wilcoxon sign rank test (p < 0.05). Mean +/- SD viscosity (cP) was significantly higher (p = 0.011) in SF obtained from the intact stifle (450 +/- 604.1) than injured (54.8 +/- 60.8) prior to surgery. Mean +/- SD HA concentrations (ug/ml) were significantly higher (p = 0.02) in synovial fluid obtained from the injured stifles (281.4 +/- 145.9) than intact stifles (141.6 +/- 132.5). No significant difference was noted within or between treatment groups in SF viscosity, HA concentrations, or serum GAG concentrations at any time following surgery. Stifles with cranial cruciate ligament insufficiency had significant alterations in SF viscosity and HA concentrations.

Intramedullary interlocking nail stabilisation of 21 humeral fractures in 19 dogs and one cat.

OBJECTIVE: To assess the suitability of the intramedullary interlocking nail to stabilise humeral diaphyseal fractures in dogs and cats. METHOD: This multi-centre study retrospectively examined medical records, between June 1994 and May 2001, of 19 dogs and one cat, in which a total of 21 humeral fractures were stabilised with intramedullary interlocking nails. RESULTS: Animals ranged in body-weight from 4 to 97 kg. Eighteen (86%) of the fractures were comminuted. Adjunctive stabilisation was used in twelve (57%) fractures and bone grafts in nine (43%) fractures. A rapid return of function was noted in the majority of animals, with 14 (67%) having good or excellent function within four days of surgery. In two fractures the repair collapsed when a single proximal transcortical screw was placed cranial to the tricipital line of the humerus. This suggests that if a single transcortical screw is placed proximally the screw should be distal or caudal to the tricipital line in order to engage sufficient cortical bone. Eighteen (86%) of the fractures healed when stabilised with intramedullary interlocking nails. Three fractures did not heal. One was in a dog where a pathological fracture was temporarily stabilised with an intramedullary interlocking nail, one in a dog that died of an abdominal crisis three weeks after surgery and one in a dog in which fracture stabilisation collapsed due to incorrect implant selection. CONCLUSION: Intramedullary interlocking nails are well suited to the stabilisation of humeral diaphyseal fractures in dogs and cats.

An unusual intronic mutation in the CYBB gene giving rise to chronic granulomatous disease.

The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5' splice site and results in multiple abnormal mRNA products.

A second case of somatic triple mosaicism in the CYBB gene causing chronic granulomatous disease.

The most common form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene that is carried on the X-chromosome and give rise to the X-linked form of the disease. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, the catalytic core of the superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene or, less frequently, at the intron/exon borders. In addition, there are large, often multi-gene, deletions. Four mutations have also been found in the promoter regions. In contrast, very few intronic mutations have been reported. Here we describe an unusual intronic mutation that causes CGD. The mutation is the insertion of 12 bp in intron XI, accompanied by the deletion of exon 12. Remarkably, the grandmother of this patient is chimeric, carrying a normal allele, the patient's allele, and an allele with a 4-nucleotide insertion at a site adjacent to the patient's insertion, in combination with a 1.5-kb deletion within intron XI. The patient's mother carries a normal allele and the patient's allele. We propose that an initial mutational event during the grandmother's embryogenesis has undergone unsuccessful DNA repair and has resulted in two aberrant alleles, one of which has been inherited by the patient and his mother. Remarkably, in the only two kindreds that have been examined in detail where deletions originating within introns have led to CGD, both families have contained members with triple somatic mosaicism.

Axial characteristics of circular external skeletal fixator single ring constructs.

OBJECTIVE: Evaluate the effects of varying ring diameter, wire tension, and wire-divergence angle on the axial stiffness characteristics of circular external skeletal fixator single-ring constructs. Study Design-Biomechanical evaluation using circular fixator components and a Delrin cylinder bone model. METHODS: Single ring constructs using two 1.6 mm diameter Kirschner wires to secure a 19 mm Delrin cylinder centered within the ring were examined. Component variables evaluated were ring diameter (50 mm, 66 mm, 84 mm, and 118 mm), wire-divergence angle (30 degrees, 60 degrees, and 90 degrees ), and wire tension (0 kg, 30 kg, 60 kg, and 90 kg). A total of 48 constructs were examined. Rings were rigidly mounted on a universal testing system and the cylinder loaded in axial compression (7.4 N/s) to 220 N. Load/displacement curves were analyzed to determine the following: the displacement (mm) that occurred before the slope of each load/displacement curve became linear, the stiffness (N/mm) of the linear portion of each load/deformation curve, and the total displacement (mm) produced at maximal load. Least-squares linear regression was used to model response variables as linear functions of ring diameter, wire divergence angle, and wire tension. Three-way interactions and 2-way interactions among independent component variables were evaluated first in the modeling process and included in a best model if response variables were found to have statistically significant regression coefficients. The regression coefficients and corresponding standard errors and covariances were used to estimate the maximal effect and standard error attributable to wire divergency angle (change from 30 degrees to 90 degrees ) and wire tension (change from 0 to 90 kg) for each ring diameter. RESULTS: All load/deformation curves had an initial exponential increase in stiffness, with the slope becoming linear at higher loads. The exponential phase was more pronounced in larger-diameter ring constructs and was mitigated by tensioning the wires. Ring diameter had the greatest influence on displacement that occurred before the curve became linear (semipartial r(2) [sp-r2] = .89), stiffness (sp-r2 = .94), and total displacement (sp-r2 = .93). Wire tension exerted a smaller influence on displacement that occurred before the curve became linear (sp-r2 =.06), stiffness (sp-r2 = .03), and total displacement (sp-r2 = .05). Wire divergence angle had a nominal effect on displacement that occurred before the curve became linear (sp-r2 = .0001), on stiffness (sp-r2 = .004), and on total displacement (sp-r2 =.003). CONCLUSIONS: Ring diameter had a profound effect on the axial stiffness characteristic of single ring constructs. Tensioning of the fixation wires can improve the axial stiffness characteristics of these constructs, particularly in larger diameter ring constructs, by mitigating the initial exponential phase of the load/deformation curve. Wire divergence angle had only a nominal differential effect on axial stability. CLINICAL RELEVANCE: Understanding how individual component variables and their interactions influence bone segment stability should help surgeons to optimize interfragmentary strain. Tensioning fixation wires is probably unnecessary in 50 mm diameter ring constructs, but assumes greater importance as ring diameter increases.

Effects of ring diameter and wire tension on the axial biomechanics of four-ring circular external skeletal fixator constructs.

OBJECTIVE: To determine relative effects of ring diameter and wire tension on axial biomechanical properties of 4-ring circular external skeletal fixator constructs. SAMPLE POPULATION: 4-ring circular external skeletal fixator constructs and artificial bone models. PROCEDURE: 4-ring constructs were assembled, using 50-, 66-, 84-, or 118-mm-diameter rings. Two 1.6-mm-diameter fixation wires were attached to opposing surfaces of each ring at intersection angles of 90 degrees and placed through a gap-fracture bone model. Three examples of each construct were loaded in axial compression at 7 N/s to a maximum load of 400 N at each of 4 wire tensions (0, 30, 60, and 90 kg). Response variables were determined from resulting load-displacement curves (construct stiffness, load at 1 mm of displacement, displacement at 400 N). RESULTS: Ring diameter and wire tension had a significant effect on all response variables and had a significant interaction for construct stiffness and displacement at 400 N. Significant differences within all response variables were seen among all 4 ring diameters and all 4 wire tensions. As ring diameter increased, effect of increasing wire tension on gap stiffness and gap displacement at 400 N decreased. Ring diameter had a greater effect than wire tension on all response variables. CONCLUSIONS AND CLINICAL RELEVANCE: Although effects of wire tension decrease as ring diameter increases, placing tension on wires in larger ring constructs is important because these constructs are inherently less stiff. The differential contribution of ring diameter, wire tension, and their interactions must be considered when using circular external skeletal fixators.

Comparison of the radius of curvature of the ulnar trochlear notch of Rottweilers and Greyhounds.

OBJECTIVE: To compare radius of curvature along the ulnar trochlear notch of Rottweilers and Greyhounds to determine whether morphologic differences exist that may contribute to the cause and pathogenesis of fragmented coronoid process in Rottweilers. SAMPLE POPULATION: Paired elbow joints from 13 Rottweilers and 14 Greyhounds. PROCEDURE: Elbow joints were radiographically scored on the basis of severity of osteoarthritic lesions. The articular contour of each ulnar trochlear notch was digitized. The radius of curvature at defined points along the ulnar trochlear notch was compared between breeds. RESULTS: Radius of curvature of the ulnar trochlear notch was not a constant function of arc length in either breed but had a consistent characteristic appearance in both breeds. Radius of curvature was greatest at each end of the ulnar trochlear notch and had 2 peaks in the midportion of the notch in both breeds. These peaks occurred farther distally in the notch and were larger in Rottweiler ulnae than Greyhound ulnae. A significant difference in mean radius of curvature was detected between breeds at these peaks. Greyhounds had significantly greater mean radius of curvature at the end of the medial coronoid process, compared with Rottweilers. CONCLUSIONS AND CLINICAL RELEVANCE: Radius of curvature of the ulnar trochlear notch is a complex function of arc length in Rottweilers and Greyhounds. The waveform has a consistent characteristic appearance in both breeds. Although significant differences were identified between breeds, associations between these differences and cause or pathogenesis of fragmented coronoid process in Rottweilers were not apparent.

Recent advances in external skeletal fixation.

While the use of external skeletal fixation was once associated with substantial postoperative morbidity, clinical and experimental studies have led to technological advances and modifications in application techniques that have greatly improved the results obtained with this treatment modality. The past decade saw numerous advances in external skeletal fixator implants, components and instrumentation, including improvements in fixation pin design, and the development of new linear external skeletal fixation systems and economical circular external skeletal fixation systems specifically engineered for use in dogs and cats. In addition, a greater understanding of fixator biomechanics and the pathobiology of the bone-fixation pin interface have improved fixator application practices. This article reviews many of the more significant recent advances in external skeletal fixation.

Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558.

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47(PHOX), p67(PHOX), p40(PHOX), and Rac1 or Rac2, with the membrane-bound cytochrome b(558). Whereas the interaction of p47(PHOX) with cytochrome b(558) is well established, an interaction between p67(PHOX) and cytochrome b(558) has never been investigated. We report here a direct interaction between p67(PHOX) and cytochrome b(558). First, labeled p67(PHOX) recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b(558)-deficient patient. Second, p67(PHOX) binds to cytochrome b(558) that has been bound to nitrocellulose. Third, GTP-p67(PHOX) bound to glutathione agarose is able to pull down cytochrome b(558.) Rac1-GTP or Rac1-GDP increased the binding of p67(PHOX) to cytochrome b(558), suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67(PHOX) and cytochrome b(558).

Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of 4 genes encoding phagocyte NADPH oxidase subunits. Unlike other CGD subtypes, in which there is great heterogeneity among mutations, 97% of affected alleles in patients previously reported with A47(0) CGD carry a single mutation, a GT deletion (DeltaGT) in exon 2 of the p47-phox gene, NCF-1. This unusually high incidence results from recombination events between NCF-1 and its highly homologous pseudogenes, in which DeltaGT originates. In 50 consecutive patients with A47(0) CGD, 4 were identified who were heterozygous for DeltaGT in NCF-1, and for the first time, 2 were identified whose DNA appeared normal at this position. To avoid co-amplification of pseudogene sequence and to enable the identification of mutations in these patients, allele-specific polymerase chain reaction was used to amplify alleles not containing DeltaGT. In each of the 4 patients who were heterozygous for DeltaGT, an additional novel mutation was identified. These were 2 missense mutations, G125 --> A in exon 2 (predicting Arg42 --> Gln) and G784 --> A in exon 8 (Gly262 --> Ser), and 2 splice junction mutations at the 5' end of intron 1, gt --> at and gtg --> gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125 --> A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574 --> A, which predicts Gly192 --> Ser but is likely to result in defective splicing because it represents the final nucleotide of exon 6.

Definition and determination of acetabular component orientation in cemented total hip arthroplasty.

OBJECTIVE: To describe the spatial orientation of the cemented acetabular component in cemented total hip arthroplasty, based on a ventrodorsal and lateral radiographic projection of the pelvis. METHODS: Equations were derived by using trigonometric relationships that describe the radiographic rotation about the longitudinal pelvic axis (alpha), transverse pelvic axis (beta), acetabular inclination (phi), acetabular inclination corrected for longitudinal pelvic rotation, version (phiC), acetabular version (theta), acetabular version corrected for longitudinal pelvic rotation (thetaC), acetabular inclination corrected for transverse pelvic rotation (phi(beta)), and acetabular version corrected for transverse pelvic rotation (theta(beta)) RESULTS: Alpha was calculated by using the equation alpha = sin(-1) (x/y) where x is the transverse distance between the dorsal spinous processes and the center of the pubis on a ventrodorsal radiograph and y is the distance from the pubis to the dorsal aspect of the first coccygeal vertebra perpendicular to the long axis of the pelvis on a lateral radiograph. Phi was calculated from the long axis (LA) and short axis (SA) of the ellipse formed by the radiopaque acetabular marker ring by using the equation phi = sin(-1) (SA/LA). phiC was calculated by using the equation phiC = phi +/- (alpha - tan(-1) (tan alpha cos thetaC)). Theta was determined as previously described. ThetaC was calculated by using the equation thetaC = tan(-1) (tan theta cos alpha). Theta(beta) and theta(beta) were calculated with the equations phi(beta) = tan(-1) (tan theta cos beta) and theta(beta) = theta - tan(-1) (sin beta), respectively. Beta could not be accurately determined from ventrodorsal and lateral pelvic radiographs. CONCLUSIONS AND CLINICAL RELEVANCE: These techniques allow for more accurate postoperative radiographic assessment of acetabular component positioning. This information can then be used in retrospective or prospective analyses examining that effects of implant positioning on clinical outcome.

Acetabular component orientation as an indicator of implant luxation in cemented total hip arthroplasty.

OBJECTIVE: To determine the sensitivity and specificity with which acetabular component angles of inclination and version could be used, alone or in combination, to predict luxation of cemented total hip arthroplasties (THA). STUDY DESIGN: Comparison of retrospectively selected cases and controls SAMPLE POPULATION: All THA performed at the University of Florida between 1991 and 1998 with the BioMedtrix system and for which at least 2 months of radiographic follow-up were available. All THA performed at the University of Georgia with the BioMedtrix system which subsequently luxated. METHODS: Acetabular component inclination angle (IA) and acetabular version angle (VA) were determined for each THA. Data were grouped according to outcome - luxation or no luxation - with the luxated cases from the 2 institutions pooled. Receiver operator characteristic (ROC) analysis was used to evaluate decision rules for using IA and VA as tests for detecting postoperative luxation. Sensitivity and specificity for luxation and 95% confidence bounds were computed with selected values of IA and VA as cut-points. RESULTS: The nonluxation group consisted of 68 THA with a median follow-up time of 5 months (range, 2-60 months). The luxation group consisted of 12 THA with a mean time to luxation of 36 days. The nonluxation group had a mean +/- standard deviation (SD) IA and VA of 40.3 degrees +/- 8.9 degrees and 71.1 +/- 13.6 degrees, respectively, whereas the luxation group had a mean +/- SD IA and VA of 34.7 degrees +/- 12.6 degrees and 72.9 degrees +/- 16.6 degrees, respectively. An IA cut-point of 37.8 degrees achieved 58.3% sensitivity and 57.4% specificity. A VA cut-point of 73 degrees achieved 75.0% sensitivity and 51.5% specificity. IA and VA considered simultaneously achieved a 50.0% sensitivity and 88.2% specificity. CONCLUSIONS AND CLINICAL RELEVANCE: ROC analysis indicated that both IA and VA considered individually or simultaneously were poor indicators of luxation. Although extreme values of IA may predict luxation with high specificity, the potential for luxation cannot be excluded based on apparently appropriate values of IA and VA. The results of this study also indicate that a successful outcome is possible with a wide range of acetabular component positions.

Molecular analysis of 9 new families with chronic granulomatous disease caused by mutations in CYBA, the gene encoding p22(phox).

Chronic granulomatous disease is a rare inherited disorder caused by nonexistent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for generating the superoxide, NADPH oxidase, involves at least 5 protein components. The absence of, or a defect in, any 1 of 4 of these proteins (p22(phox), p47(phox), p67(phox), or gp91(phox)) gives rise to the known types of chronic granulomatous disease. One of the rarest forms of the disease is due to defects in the CYBA gene encoding p22(phox), which together with gp91(phox) forms flavocytochrome b(558), the catalytic core of NADPH oxidase. To date, only 9 kindreds with p22(phox) deficiency have been described in the literature comprising 10 mutant alleles. Four polymorphisms in the CYBA gene have also been reported. Here we describe 9 new, unrelated kindreds containing 12 mutations, 9 of which are novel. In addition, we report 3 new polymorphisms. The novel mutations are (a) deletion of exons 2 and 3, (b) a missense mutation in exon 3 (T155-->C), (c) a splice site mutation at the 5' end of intron 3, (d) a missense mutation in exon 2 (G74-->T), (e) a nonsense mutation in exon 1 (G26-->A), (f) a missense mutation in exon 4 (C268-->T), (g) a frameshift in exon 3 due to the insertion of C at C162, (h) a nonsense mutation in exon 2 (G107-->A), and (i) a missense mutation in exon 2 (G70-->A).

p40(phox) Participates in the activation of NADPH oxidase by increasing the affinity of p47(phox) for flavocytochrome b(558).

NADPH oxidase is one of the major components of the innate immune system and is used by phagocytes to generate microbicidal reactive oxygen species. Activation of the enzyme requires the participation of a minimum of five proteins, p22(phox), gp91(phox) (together forming flavocytochrome b(558)), p47(phox), p67(phox) and the GTP-binding protein, Rac2. A sixth protein, p40(phox), has been implicated in the control of the activity of NADPH oxidase principally based on its sequence homology to, and physical association with, other phox components, and also the observation that it is phosphorylated during neutrophil activation. However, to date its role in regulating the activity of the enzyme has remained obscure, with evidence for both positive and negative influences on oxidase activity having being reported. Data are presented here using the cell-free system for NADPH oxidase activation that shows that p40(phox) can function to promote oxidase activation by increasing the affinity of p47(phox) for the enzyme approx. 3-fold.

NADH-ferric reductase activity associated with dihydropteridine reductase.

In mammals dietary ferric iron is reduced to ferrous iron for more efficient absorption by the intestine. Analysis of a pig duodenal membrane fraction revealed two NADH-dependent ferric reductase activities, one associated with a b-type cytochrome and the other not. Purification and characterization of the non-cytochrome ferric reductase identified a 31 kDa protein. MALDI-MS analysis and amino acid sequencing identified the ferric reductase as being related to the 26 kDa liver NADH-dependent quinoid dihydropteridine reductase (DHPR). The NADH-dependent DHPR ferric reductase activity was found to be pteridine-independent since exhaustive dialysis did not reduce activity and heat-inactivation destroyed activity. In intestinal Caco-2 cells, DHPR mRNA levels were found to be regulated by iron. Thus, DHPR appears to be a dual function enzyme, a NADH-dependent dihydopteridine reductase and an iron-regulated, NADH-dependent, pteridine-independent ferric reductase.