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Biomedical subjects

J B Boyd

Publications and source records attributed to J B Boyd.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos.

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.

Animals↗
PubMed1992

Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis.

Animals↗
PubMed1992

Nuclease modification in Chinese hamster cells hypersensitive to DNA cross-linking agents--a model for Fanconi anemia.

Fanconi anemia is a human inherited disease that is characterized by cellular hypersensitivity to DNA cross-linking agents. A number of potential experimental models for that disorder have been developed by selecting mutants that are hypersensitive to bifunctional mutagens. The six mutants of that class in Drosophila, all of which map to the mus308 locus, express an alteration in a mitochondrial nuclease. A recent extension of that observation to cell lines from complementation group A of Fanconi anemia has established a new cellular phenotype for that disorder. In the current study an analogous enzyme has been analyzed in eight recently isolated Chinese hamster cell lines that are hypersensitive to cross-linking agents. Among these lines. V-H4 and V-B7 are shown to exhibit an enzyme modification analogous to that observed in the mutant Drosophila and human cells. These results validate the nuclease assay as an indicator of the Fanconi defect and further establish the V-H4 cell line as a valuable cellular model for analysis of the Fanconi A defect.

Animals↗
PubMed1992

Massive pharyngocutaneous fistulae: salvage with two-layer flap closure.

Massive pharyngocutaneous fistulae may be arbitrarily defined as those involving two-thirds or more of the circumference of the pharyngeal wall. Three such patients are presented, all after failed radiotherapy and surgery. The principles of management consist of salivary diversion, complete debridement, nutritional support, prophylactic antibiotics, and two-layer, well-vascularized overlapping closure. We currently recommend a radial forearm flap used in conjunction with a pectoralis muscle (or musculocutaneous) flap for rapid rehabilitation of these patients, particularly in the presence of obesity or an irradiated bed.

Aged↗
PubMed1992

Augmentation of skeletal muscle survival in the latissimus dorsi porcine model using acute ischemic preconditioning.

Reconstruction of head and neck defects commonly involve local and distal muscle transplants. Ischemic flap necrosis is a well recognized complication of autogenous muscle transplants. Research in the field of myocardial survival has recently shown that repeated brief periods of ischemia followed by reperfusion result in improve muscle survival when the cardiac muscle is subsequently subjected to prolonged ischemia. This is called preconditioning and the mechanism is unknown. The concept is discussed in this paper and presented as a potential new, non-pharmacological strategy to reduce skeletal muscle ischemic necrosis. A review of the development and application of preconditioning in myocardial muscle survival is presented, and causes of ischemic muscle necrosis are discussed. Potential mechanisms of preconditioning in muscle are also presented. The extrapolation of the concept of preconditioning is discussed with an examination of a potential experimental model to test this concept: the latissimus dorsi flap in the pig. The results of our investigations into the effect of preconditioning on skeletal muscle and its possible mechanism are discussed. Preconditioning may prove to be a useful method to improve skeletal muscle tolerance to sustained normothermic global ischemia. This may have important clinical benefits in muscle transplantation. To our knowledge, this is the first publication to discuss the application of preconditioning in skeletal muscle.

Animals↗
PubMed1991

Mechanism of action of deoxyribonuclease II from human lymphoblasts.

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme. Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I.

Base Sequence↗
PubMed1991

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.

Cell Fractionation↗
PubMed1991

P transposition in Drosophila provides a new tool for analyzing postreplication repair and double-strand break repair.

A genetic screen has been developed in Drosophila for identifying host-repair genes responsible for processing DNA lesions formed during mobilization of P transposable elements. Application of that approach to repair deficient mutants has revealed that the mei-41 and mus302 genes are necessary for recovery of P-bearing chromosomes undergoing transposition. Both of these genes are required for normal postreplication repair. Mutants deficient in excision repair, on the other hand, have no detected effect on the repair of transposition-induced lesions. These observations suggest that P element-induced lesions are repaired by a postreplication pathway of DNA repair. The data further support recent studies implicating double-strand DNA breaks as intermediates in P transposition, because the mei-41 gene has been genetically and cytologically associated with the repair of interrupted chromosomes. Analysis of this system has also revealed a striking stimulation of site-specific gene conversion and recombination by P transposition. This result strongly suggests that postreplication repair in this model eukaryote operates through a conversion/recombination mechanism. Our results also support a recently developed model for a conversion-like mechanism of P transposition (Engels et al., 1990). Involvement of the mei-41 and mus302 genes in the repair of P element-induced double-strand breaks and postreplication repair points to a commonality in the mechanisms of these processes.

Animals↗
PubMed1991

Penile replantation.

Amputation of the penis is thankfully an uncommon injury. Because of its rarity, a uniform plan of management to deal with this difficult injury has yet to be agreed on. We describe a man who sustained such an injury and his subsequent clinical course. The relevant history and pertinent anatomy is highlighted. Additionally, a systematic approach to dealing with this devastating injury is presented.

Amputation, Traumatic↗
PubMed1991

Augmentation of transmidline skin perfusion and viability in transverse rectus abdominis myocutaneous (TRAM) flaps in the pig.

The aim of this experiment was to design a clinically relevant TRAM flap in the pig and to use this flap model to study the effectiveness of preoperative ligation of the dominant vascular pedicle in augmentation of muscle and skin capillary blood flow and skin viability in the TRAM flap. This TRAM flap model was based on the deep inferior epigastric vascular pedicle, with the center of the transverse skin paddle attached to the underlying rectus abdominis muscle at the superior end of the muscle and extending bilaterally from its attached muscle. The transverse skin paddle (8 x 30 cm) included a contralateral and ipsilateral random portion of skin. This flap model was based on the deep inferior epigastric rather than the superior epigastric vascular pedicle because the deep inferior epigastric vascular pedicle is the smaller of the two in the pig and augmentation of its blood supply by ligation of the dominant superior epigastric vascular pedicle resembles more closely the clinical situation. It was observed that ligation of the dominant superior epigastric vascular pedicle 14 days prior to raising the TRAM flap significantly (p less than 0.05; n = 5) increased the total muscle and skin capillary blood flow and skin viability in the transverse skin paddle compared with the sham-operated control (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Abdominal Muscles↗
PubMed1991

[Vascular territory expansion: a surgical technique for augmenting survival of an animal model of a musculocutaneous flap].

Ischemia-induced flap failure remains a major concern for reconstructive surgeons. A porcine rectus abdominis musculocutaneous (TRAM) flap was designed and subsequently used to study our hypothesis of vascular territory expansion whereas the perfusion territory of a previously ligated vessel is taken over by the remaining vessel(s). Specifically, we observed that ligation of the superior epigastric (SE) vessels two weeks prior to raising the TRAM flap significantly (p less than 0.05) increased the length and area of the viable skin in the transverse skin paddles of the treatment flaps compared to the contralateral sham manipulated control flaps. This significant increase in skin viability was seen to be accompanied by a significant (p less than 0.05) increase in skin and muscle capillary blood flow in the treated flaps compared with the controls (n = 9). The mechanism of vascular territory expansion is unclear. We postulate that hypoxia resulting from the ligation of the SE vessels prior to the flap surgery may play a role in triggering the deep inferior epigastric (DIE) artery to take over some of the territory previously perfused by the SE artery.

Abdominal Muscles↗
PubMed1990

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents.

The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents. Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pI of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg++ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50-100 mM. Nuclease 3 exhibits a temperature optimum of 42 degrees C and is insensitive to N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster.

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.

Alleles↗
PubMed1990

X-ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster.

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.

Animals↗
PubMed1990

The iliac crest and the radial forearm flap in vascularized oromandibular reconstruction.

Sixty cases (59 patients) of oromandibular reconstruction using vascularized iliac crests were compared with 13 in which radial osteocutaneous flaps were used. These patients were reviewed from the standpoint of cosmetic results and function as well as their operative and postoperative courses. In both groups, the results were generally good. However, revisionary surgery was more frequent in those receiving the iliac crest. This group also had a higher incidence of intraoral wound breakdown and bone exposure. Nevertheless, the sheer size of the iliac crest made it ideal for massive oromandibular defects, just as its natural curvature lent itself to precise replication of the mandible in bone-only reconstructions. Its bulk proved a major obstacle in small composite defects. The radial forearm flap carried thin, pliable, well-vascularized skin that was superior to groin skin for oral lining. Bone gaps of up to 9 cm could be handled with ease, thus making it complementary to the iliac crest over the wide spectrum of mandibular reconstruction.

Adolescent↗
PubMed1990

mus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease.

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.

Animals↗
PubMed1990

Surgical augmentation of skin blood flow and viability in a pig musculocutaneous flap model.

A porcine rectus abdominis musculocutaneous (TRAM) flap model was designed and validated in nine pigs. This TRAM flap was based on the deep inferior epigastric (DIE) vessels with an 8 x 18 cm transverse skin paddle at the superior end of the rectus abdominis muscle. The model was subsequently used to test our hypothesis of surgical augmentation of flap viability by vascular territory expansion. Specifically, we observed that ligation of the superior epigastric (SE) vessels at 4, 7, 14, and 28 days (N = 6 to 8) prior to raising the TRAM flaps significantly increased (p less than 0.05) the length and area of the viable skin in the transverse skin paddles of the treatment flaps compared with the contralateral shammanipulated control flaps. This significant increase in skin viability was seen to be accompanied by a significant increase (p less than 0.05) in skin and muscle capillary blood flow in the treatment TRAM flaps compared with the controls (N = 9). The mechanism of vascular territory expansion is unclear. We postulate that hypoxia resulting from the ligation of the superior epigastric vessels prior to the flap surgery may play a role in the triggering of the deep inferior epigastric artery to take over some of the territory previously perfused by the superior epigastric artery. This would then increase the skin and muscle capillary blood flow in the transverse paddle when the TRAM flap was raised on the deep inferior epigastric vascular pedicle.

Animals↗