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Biomedical subjects

A R Cross

Publications and source records attributed to A R Cross.

At least 19 recordsLinked to original sources
PubMed2005

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.

Adjuvants, Immunologic↗
PubMed2002

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.

Animals↗
PubMed2001

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.

B-Lymphocytes↗
PubMed2001

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).

Cytochrome b Group↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Child, Preschool↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

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).

Adolescent↗
PubMed2000

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.

Cell Membrane↗
PubMed2000

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.

Animals↗
PubMed1999

Functional analysis of NADPH oxidase in granulocytic cells expressing a delta488-497 gp91(phox) deletion mutant.

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O(2)(-)) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91(phox), the large subunit of the flavocytochrome b(558) heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (delta488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant triangle delta488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional delta488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to delta488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O(2)(-) production in the cell-free oxidase assay (IC50, approximately 500 micromol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant delta488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the delta488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.

Cell Differentiation↗
PubMed1999

A novel mutation in the CYBB gene resulting in an unexpected pattern of exon skipping and chronic granulomatous disease.

Chronic granulomatous disease is a rare inherited disorder caused by non-existent 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 superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.

Base Sequence↗
PubMed1999

The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex.

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.

Animals↗
PubMed1999

Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.

We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations.

Anions↗
PubMed1999

Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations.

The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120-150 mol of O2(-)/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.

Cell Membrane↗