Ambulatory care in interventional radiology: a framework for radiology day-case.

AIM: To describe a governance framework for setting up an ambulatory care unit in the interventional radiology setting. MATERIALS AND METHODS: Guidance from NHS England, Getting it right first time, The kings fund, NHS modernisation agency, NHS Improvement, The General Medical Council, The Royal college of Radiologists, The British society of interventional radiology, The Care Quality Commission, and the British Association of Day Surgery was reviewed and referenced as evidence for the governance pathway for day-case patients. RESULTS: A complete pathway for ambulatory care of patients in interventional radiology from referral to discharge is outlined with a discussion of examples of quality and safety. CONCLUSION: Successful implementation of an ambulatory care unit in interventional radiology requires a collaborative, multidisciplinary approach that links in with the NHS improvements ethos for more day-case procedures.

Sacral osteochondrosis in two German Shepherd Dogs.

Two young adult male castrated German Shepherd Dogs were referred for evaluation of intermittent episodes of hindlimb pain. Physical examination suggested lumbosacral stenosis, and plain radiographs and computed tomography revealed lesions consistent with sacral osteochondrosis. One dog had osteochondral fragments removed surgically; the other was managed conservatively. The surgically treated dog had complete resolution of clinical signs whereas the dog managed conservatively had repeated episodes of mild pain and received one short course of non-steroidal anti-inflammatory medication in 18 months. Sacral osteochondrosis has not been previously reported in Australia.

Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia.

Friedreich ataxia (FRDA) is caused by hyperexpansion of GAA*TTC repeats located in the first intron of the FXN gene, which inhibits transcription leading to the deficiency of frataxin. The FXN gene is an excellent target for therapeutic intervention since (i) 98% of patients carry the same type of mutation, (ii) the mutation is intronic, thus leaving the FXN coding sequence unaffected and (iii) heterozygous GAA*TTC expansion carriers with approximately 50% decrease of the frataxin are asymptomatic. The discovery of therapeutic strategies for FRDA is hampered by a lack of appropriate molecular models of the disease. Herein, we present the development of a new cell line as a molecular model of FRDA by inserting 560 GAA*TTC repeats into an intron of a GFP reporter minigene. The GFP_(GAA*TTC)(560) minigene recapitulates the molecular hallmarks of the mutated FXN gene, i.e. inhibition of transcription of the reporter gene, decreased levels of the reporter protein and hypoacetylation and hypermethylation of histones in the vicinity of the repeats. Additionally, selected histone deacetylase inhibitors, known to stimulate the FXN gene expression, increase the expression of the GFP_(GAA*TTC)(560) reporter. This FRDA model can be adapted to high-throughput analyses in a search for new therapeutics for the disease.

Pediatric hospitalists: quality care for the underserved?

Despite the rapid growth in pediatric hospitalist services, there is little empiric information about the impact of pediatric hospitalists. This study compared process and outcome variables related to the inpatient care of 182 pediatric patients, half of whom were cared for by hospitalists and half by their primary care providers (PCP). Results indicated that, while hospitalists cared for patients of substantially lower socioeconomic status, they delivered care more economically for patients with asthma, with no significant differences in rates of return to the emergency room or rehospitalizations. Children in both services demonstrated equivalent levels of returning to their PCP for follow-up visits and were in equally good health 1 month after discharge. Additionally, no negative impact was evident on patient satisfaction at discharge; in fact, the hospitalists' patients were more satisfied with aspects of their care. Hospitalists may, therefore, provide a vital service by ensuring quality inpatient care for low-income children.

Identification of aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation.

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.

Sticky DNA, a self-associated complex formed at long GAA*TTC repeats in intron 1 of the frataxin gene, inhibits transcription.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the expansion of GAA.TTC repeats in the first intron of the frataxin (X25) gene. FRDA patients carrying two expanded GAA.TTC repeats show very low levels of mature frataxin mRNA and protein. A novel type of unusual DNA structure, sticky DNA, was previously found in the expanded GAA.TTC repeats from FRDA patients. To evaluate the effect of sticky DNA on transcription, in vitro transcription studies of (GAA.TTC)(n) repeats (where n = 9-150) were carried out using T7 or SP6 RNA polymerase. When a gel-isolated sticky DNA template was transcribed, the amount of full-length RNA synthesized was significantly reduced compared with the transcription of the linear template. Surprisingly, transcriptional inhibition was observed not only for the sticky DNA template but also another DNA molecule used as an internal control in an orientation-independent manner. The molecular mechanism of transcriptional inhibition by sticky DNA was a sequestration of the RNA polymerases by direct binding to the complex DNA structure. Moreover, plasmids containing the (GAAGGA.TCCTTC)(65) repeat, which does not form sticky DNA, did not inhibit in vitro transcription, as expected. These results suggest that the role of sticky DNA in FRDA may be the sequestration of transcription factors.

Recombinant human DNA (cytosine-5) methyltransferase. III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG.CCG sequence.

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-l-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-l-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded CGG.CCG triplet repeat sequence.

GGA*TCC-interrupted triplets in long GAA*TTC repeats inhibit the formation of triplex and sticky DNA structures, alleviate transcription inhibition, and reduce genetic instabilities.

Large expansions of GAA.TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA). Sticky DNA, based on R.R.Y triplexes, was found at the expanded GAA.TTC repeats from FRDA patients. The (GAAGGA.TCCTTC)(65) repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA. To elucidate the behavior of sticky DNA, we introduced various extents of GGA.TCC interruptions into the long GAA.TTC repeat. More than 20% of GGA.TCC interruptions abolished the formation of sticky DNA. However, the GAA.TTC repeats with less than 11% of GGA.TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence. These triplexes showed different P1 nuclease sensitivities, and the GGA.TCC interruptions were slightly more sensitive than the surrounding GAA.TTC repeats. Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA.TTC tracts. Furthermore, the greater the extent of interruptions of the GAA.TTC repeats, the less inhibition of in vitro transcription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA. We propose that the interruptions introduce base mismatches into the R.R.Y triplex, which explains the observed chemical and biological properties.

Pkd1 unusual DNA conformations are recognized by nucleotide excision repair.

The 2.5-kilobase pair poly(purine.pyrimidine) (poly(R.Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3). The full-length poly(R.Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R.Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R.Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid. These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.

The intrinsically unstable life of DNA triplet repeats associated with human hereditary disorders.

Expansions of specific DNA triplet repeats are the cause of an increasing number of hereditary neurological disorders in humans. In some diseases, such as Huntington's and several spinocerebellar ataxias, the repetitive DNA sequences are translated into long tracts of the same amino acid (usually glutamine), which alters interactions with cellular constituents and leads to the development of disease. For other disorders, including common genetic disorders such as myotonic dystrophy and fragile X syndrome, the DNA repeat is located in noncoding regions of transcribed sequences and disease is probably caused by altered gene expression. In studies in lower organisms, mammalian cells, and transgenic mice, high frequencies of length changes (increases and decreases) occur in long DNA triplet repeats. These observations are similar to other types of repetitive DNA sequences, which also undergo frequent length changes at genomic loci. A variety of processes acting on DNA influence the genetic stability of DNA triplet repeats, including replication, recombination, repair, and transcription. It is not yet known how these different multienzyme systems interact to produce the genetic mutation of expanded repeats. In vitro studies have identified that DNA triplet repeats can adopt several unusual DNA structures, including hairpins, triplexes, quadruplexes, slipped structures, and highly flexible and writhed helices. The formation of stable unusual structures within the cell is likely to disturb DNA metabolism and be a critical intermediate in the molecular mechanism(s) leading to genetic instabilities of DNA repeats and, hence, to disease pathogenesis.

Chromatin structure of yeast minichromosomes containing triplet repeat sequences associated with human hereditary neurological diseases.

Expansion of triplet repeat sequences such as (CTG)n, (CGG)n, and (GAA)n causes human genetic diseases. Since DNA is packaged into arrays of nucleosomes in eukaryotic cells, chromatin may be involved in the mechanism of triplet repeat diseases. To elucidate this issue, we have examined effects of triplet repeat sequences on the chromatin organization in vivo using well defined yeast minichromosomes. We show here that (CGG)12 disrupts an array of positioned nucleosomes, whereas (CTG)12 promotes the nucleosome formation. Thus, triplet repeat sequences can affect the chromatin organization in vivo, which may contribute to the triplet repeat expansion or alterations in the expression of genes associated with triplet repeat diseases.

Gene conversion (recombination) mediates expansions of CTG[middle dot]CAG repeats.

Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468-23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.

Tandem duplication. A novel type of triplet repeat instability.

Triplet repeat sequence (TRS) inserts containing (CTG.CAG)(n) (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli. The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence. The duplication process required the presence of two or more TRS-containing units. Close proximity (170 base pairs) of the TRS to the R6K gamma origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process. These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication. This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.

Length of CTG.CAG repeats determines the influence of mismatch repair on genetic instability.

We showed previously that mutations in methyl-directed mismatch repair of Escherichia coli reduced the occurrence of large deletions in (CTG.CAG)(175) repeats contained on plasmids. By contrast, other workers reported that mutations in mismatch repair increase the frequency of small-length changes in the shorter (CTG.CAG)(64). Using plasmids with a variety of lengths and purity of (CTG.CAG) repeats, we have resolved these apparently conflicting observations. We show that all lengths of (CTG.CAG) repeats are subject to small-length changes (eight repeats) in (CTG.CAG)(n) occur more readily in cells with active mismatch repair. The frequency of large deletions is proportional to the tract length; in our assays they become prominent in tracts greater than 100 repeats. Interruptions in repeat purity enhance the occurrence of large deletions. In addition, we observed a high level of incidence of deletions in (CTG.CAG) repeats for cultures passing repeatedly through stationary phase during long-term growth experiments of all strains (i.e. with active or inactive mismatch repair). These results agree with current theories on mismatch repair acting on DNA slippage events that occur in DNA triplet-repeats.

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Genetic instabilities of triplet repeat sequences by recombination.

The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.

A nonpathogenic GAAGGA repeat in the Friedreich gene: implications for pathogenesis.

An individual with late-onset ataxia was found to be heterozygous for an unusual (GAAGGA)65 sequence and a normal GAA repeat in the frataxin gene. No frataxin point mutation was present, excluding a form of Friedreich ataxia. (GAAGGA)65 did not have the inhibitory effect on gene expression in transfected cells shown by pathogenic GAA repeats of similar length. GAA repeats, but not (GAAGGA)65, adopt a triple helical conformation in vitro. We suggest that such a triplex structure is essential for suppression of gene expression.

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.

Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at