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Biomedical subjects

K A Vincent

Publications and source records attributed to K A Vincent.

At least 19 recordsLinked to original sources
PubMed2001

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Adenoviruses, Human↗
PubMed2000

Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O(2) homeostasis, including vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. We sought to exploit this native adaptive response to hypoxia as a treatment for chronic ischemia. METHODS AND RESULTS: A hybrid protein consisting of DNA-binding and dimerization domains from the HIF-1alpha subunit and the transactivation domain from herpes simplex virus VP16 protein was constructed to create a strong, constitutive transcriptional activator. After transfection into HeLa, C6, and Hep3B cells, this chimeric transcription factor was shown to activate expression of the endogenous VEGF gene, as well as several other HIF-1 target genes in vitro. The bioactivity of HIF-1alpha/VP16 hybrid gene transfer in vivo was examined in a rabbit model of hindlimb ischemia. Administration of HIF-1alpha/VP16 was associated with significant improvements in calf blood pressure ratio, angiographic score, resting and maximal regional blood flow, and capillary density (all P:<0.01). CONCLUSIONS: The HIF-1alpha/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.

Angiography↗
PubMed1998

Latex allergy in non-spina bifida patients: unfamiliar intra-operative anaphylaxis.

BACKGROUND: The medical literature has described the prevalence of latex allergy in the spina bifida population and its implications for surgical intervention. We report three cases of severe and unexpected intra-operative anaphylaxis secondary to latex exposure in non-spina bifida patients. METHODS: A retrospective review of case notes identified three non-spina bifida patients who suffered intra-operative anaphylaxis due to latex allergy. Personal and telephone interview and patient chart review was performed to detail a past history of multiple latex exposure, atopy, the anaphylaxis event and the postoperative outcome. RESULTS: Three non-spina bifida patients are described. One suffered a cardiopulmonary arrest, the remaining two patients had severe vascular hypotension and airway resistance that was only relieved after administration of vasoconstrictors and bronchodilators. Postoperatively, all three tested strongly positive to latex allergen testing. Each patient had a history of multiple surgical latex exposure and specific allergies or allergic-type symptoms pre-operatively. CONCLUSION: We believe that the predictors of a severe allergic reaction to latex with surgical exposure in non-spina bifida patients may be similar to those predictors known in the spina bifida population. Identification of such at-risk patients will reduce the risk of significant intra-operative morbidity and possible mortality by the introduction of a latex-free operating environment.

Abnormalities, Multiple↗
PubMed1997

Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration.

Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-beta (PDE-beta) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-beta cDNA. Following intraocular injection of AAV. PDE-beta, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.

Animals↗
PubMed1997

Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products.

Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.

Avian Sarcoma Viruses↗
PubMed1995

An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer.

Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.

Amino Acid Sequence↗
PubMed1994

Low incidence of point mutations detected in the p53 tumor suppressor gene from chemically induced rat renal mesenchymal tumors.

Previous studies have shown renal mesenchymal tumors (RMTs) induced in rats by a single intrarenal injection of nickel subsulfide and iron are more pleomorphic and metastatically aggressive than RMTs induced by a single ip injection of methyl(methoxymethyl)nitrosamine (DMN-OMe). While both RMT types contain high levels of K-ras activation, the specific mutational spectra within codon 12 of K-ras are quite different. Nickel subsulfide and iron-induced tumors exhibited codon 12 GGT-->GTT transversions exclusively, while DMN-OMe RMTs showed a wide array of codon 12 mutations, as well as mutations within codons 61 and 63 [K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. D. Reed, and A. O. Perantoni, Cancer Res., 52: 4747-4751, 1992; K. G. Higinbotham, J. M. Rice, and A. O. Perantoni, Mol. Carcinog., 5: 136-139, 1992]. In an effort to further correlate carcinogen-specific molecular events in renal tumors, we investigated the p53 tumor suppressor gene in RMTs induced by these two carcinogens for the presence of point mutations. The evolutionarily conserved portion of the coding region of the gene, including part of exon 4 through exon 10, was surveyed for point mutations utilizing single-strand conformation polymorphism and chemical cleavage of mismatches analyses. None (0 of 10) of the nickel subsulfide and iron-induced RMTs and only 1 of 10 DMN-OMe-induced tumors that were evaluated contained point mutations within this portion of the p53 gene. Direct sequencing of the one single-strand conformation polymorphism and chemical cleavage of mismatches-"positive" DMN-OMe-induced RMT revealed a GCC-->GTC (Ala-->Val) transition in codon 345 within exon 10. These results suggest that the different tumorigenic phenotypes exhibited by these two RMTs are not the result of specific mutations or patterns of mutations within the portion of the p53 gene examined and that the mutated p53 tumorigenic pathway, whereby p53 plays a major role in many human neoplasms, does not function in RMTs induced by either agent.

Animals↗
PubMed1993

The Sauve-Kapandji procedure for reconstruction of the rheumatoid distal radioulnar joint.

Our experience with the Sauve-Kapandji procedure for reconstruction of the rheumatoid distal radioulnar joint is reported. Twenty-one wrists in 17 patients were followed for an average of 39 months postoperatively. Average range of motion at follow-up evaluation was pronation to 78 degrees and supination to 86 degrees. X-ray films demonstrated that significant ulnarward and palmarward translocation of the carpus was prevented. The Sauve-Kapandji procedure provides a stable ulnar side support in the rheumatoid wrist with distal radioulnar degeneration.

Adult↗
PubMed1993

Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations.

Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.

Amino Acid Sequence↗
PubMed1992

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Base Sequence↗
PubMed1991

Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells.

A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

Animals↗
PubMed1990

Host sequences flanking the HIV provirus.

A conserved property of retroviral proviruses is the presence of a direct repeat in the host DNA immediately flanking the viral sequence; each virus generates a repeat with a characteristic length. By sequencing the viral/host DNA junctions from five HIV-1 proviral clones, we have confirmed that integration of HIV results in the generation of a five basepair direct repeat. A target sequence in uninfected host DNA was analyzed to establish that the five basepair sequence flanking the provirus was present only once prior to integration. Of the five proviruses examined, two were found to have integrated in known repetitive sequence elements of the human genome; one in a Line-1 element and a second in satellite DNA.

Base Sequence↗
PubMed1989

Evolution and transcription of old world monkey globin genes.

Although apparently inactive in the whole animal, the delta globin genes from three species of Old World monkey (rhesus, baboon and green monkey) are all functional in an in vitro transcription assay. Their activities in vitro are similar to that of the functional human delta gene. A fourth monkey gene, from the colobus monkey, is transcribed approximately fivefold less efficiently than the others. This reduced in vitro activity results from a 20 base-pair deletion, which removes the normal site of mRNA initiation. When the deletion is repaired by site-directed mutagenesis, transcriptional activity increases to the level observed for the other delta genes. We also report the complete nucleotide sequences of the colobus beta, colobus delta, and rhesus delta genes. Sequence comparisons show that the delta and beta genes in the same species have not exchanged genetic information since the divergence of the human and monkey lineages. Phylogenetic analysis of these sequences affirms that the Old World monkey delta genes are evolving more rapidly than their functional counterparts. Moreover, the rate of replacement substitutions has risen to equal that of non-coding DNA, as expected for genes no longer under selective constraint.

Amino Acid Sequence↗
PubMed1989

Intraoperative ultrasonography for reduction of thoracolumbar burst fractures.

The purpose of this study was to determine the efficacy of intraoperative ultrasound monitoring in the reduction and stabilization of thoracolumbar burst fractures of the spine. Thirty-one patients underwent a posterior approach for reduction and stabilization of a thoracolumbar burst fracture, with complete follow-up available on 26. Intraoperative real-time sonography was used for monitoring retropulsed fragments during distraction and fracture impaction. Computed tomography was used to compare preoperative canal compromise and postoperative reduction. Average canal compromise preoperatively was 66.5%, and the average canal compromise postoperatively was 18.7%. Neurologic function was not changed in 16, improved in ten, and worsened in none. Average hospital stay was 21.6 days. No complications were directly attributable to the use of the ultrasound. The authors believe that ultrasonography provides a safe and accurate method of intraoperatively evaluating reduction of burst fracture of the thoracolumbar spine from the posterior approach.

Follow-Up Studies↗
PubMed1986

Persistence or rapid generation of DNA length polymorphism at the zeta-globin locus of humans.

Extensive restriction mapping of 76 human genomic DNAs defines multiple sites of length and point mutation near the zeta-globin locus, which codes for an embryonic alpha-like globin chain. There are two major sites of DNA length variation: one in the intergenic region with three alleles and one in the first intron of the zeta 1 gene with at least four alleles. Our mapping establishes that the intronic polymorphism is associated with a tandem array of short, repeated sequences. The length alleles occur in each of four human populations sampled, suggesting an ancient origin with persistence of several length alleles, or rapid regeneration of these particular variants. Four polymorphic restriction sites were also found; the frequency of polymorphic sites is comparable to that found in the human beta-globin gene region. Analysis of haplotypes indicates either that multiple recombinations have occurred near the 5' end of the zeta 1 gene or that this region is prone to recurrent length mutation.

Alleles↗
PubMed1984

Genetic structure of natural populations of the red-spotted newt, Notophthalmus viridescens.

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I = 0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H = 0.157) and neotenic populations are only slightly, but significantly, less variable (H = 0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.

Alleles↗
PubMed1983

Rise and fall of the delta globin gene.

This paper examines the molecular basis for the origin and subsequent silencing of the delta globin gene during the evolution of higher primates. We cloned and sequenced the delta gene from representatives of the two most distantly related types of Old World monkeys in which the gene is silent. The silent delta genes of these two monkeys (rhesus and colobus) are highly homologous to each other (97.5%) and to the functional human delta gene (94%). The presumed mutation that silenced the delta gene is therefore unlikely to be obscured by later mutations at the same site. This silencing mutation could have affected transcription since we find no delta transcript in rhesus bone marrow. Furthermore, the delta globin genes from colobus and rhesus monkeys are many times less efficient than the human delta gene in a cell-free transcription assay. The two Old World monkeys share only three nucleotide substitutions in the first 100 base-pairs of the DNA sequence 5' to the site for initiation of transcription when compared to human delta. It is likely that the in vitro silencing of the delta gene was brought about by one (or a combination) of these three substitutions. Three analysis shows how the delta sequences are related to those for other beta-like genes, including the colobus beta gene whose sequence we report. One part of the delta gene appears to be derived from a (pseudo?) beta gene that diverged from beta before the divergence of mice and men. The other part is closely related to the beta gene of higher primates. The mosaic nature of the delta gene is apparently due to a partial conversion which took place between the pseudo beta and the functional beta gene before the divergence of the lineages leading to Old World monkeys and hominoids.

Animals↗