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Biomedical subjects

D A Morrison

Publications and source records attributed to D A Morrison.

At least 19 recordsLinked to original sources

Evolution of ruminant Sarcocystis (Sporozoa) parasites based on small subunit rDNA sequences.

We present an evolutionary analysis of 13 species of Sarcocystis, including 4 newly sequenced species with ruminants as their intermediate host, based on complete small subunit rDNA sequences. Those species with ruminants as their intermediate host form a well-supported clade, and there are at least two major clades within this group, one containing those species forming microcysts and with dogs as their definitive host and the other containing those species forming macrocysts and with cats as their definitive host. Those species with nonruminants as their intermediate host form the paraphyletic sister group to these clades. Most of the species have considerable genotypic differences (differing in more than 100 nucleotide positions), except for S. buffalonis and S. hirsuta. There is a large suite of genotypic differences indicating that those species infecting ruminant and nonruminant hosts have had very different evolutionary histories, and similarly for the felid- and canid-infecting species. Furthermore, the rDNA sequences that represent the different structural regions of the rRNA molecule have very different genotypic behavior within Sarcocystis. The evolution of these regions should be functionally constrained, and their differences can be explained in terms of the importance of the nucleotide sequences to their functions.

Animals

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences.

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.

Animals

The genus Hammondia is paraphyletic.

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

Animals

Comparison of the biological characteristics of two isolates of Neospora caninum.

This study compared the biological and genetic properties of a bovine (NC-SweB1) and a canine (NC-Liverpool) isolate of Neospora caninum. A mouse model for CNS infection demonstrated marked differences in pathogenicity between the isolates. NC-Liverpool induced severe clinical signs of neosporosis in 57/58 mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss. In contrast NC-SweB1 induced similar but less severe symptoms in a much smaller proportion of mice over the same time-period. Statistically significant differences were observed between the isolates in the response (mean weight loss) of mice through time to the different doses inoculated. Histopathological effects on brain tissue reflected the isolate-based differences described above. NC-Liverpool infection resulted in intense inflammatory infiltrates and highly necrotic lesions whereas NC-SweB1 induced a milder meningoencephalitis. Passage in cell-culture over a period of 14 months did not affect the pathogenicity of NC-Liverpool. Immunoblots showed that antibodies to N. caninum appeared earlier in mice inoculated with NC-Liverpool than with NC-SweB1. Finally, RAPD-PCR analysis of NC-Liverpool DNA generated profiles distinct from that observed with DNA from NC-SweB1 or Toxoplasma gondii. In summary this study provides evidence for significant biological and genetic differences between 2 isolates of N. caninum.

Animals

The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence.

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

Animals

Construction and analysis of a library for random insertional mutagenesis in Streptococcus pneumoniae: use for recovery of mutants defective in genetic transformation and for identification of essential genes.

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.

Bacillus subtilis

Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation.

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.

Bacterial Proteins

Insertion-duplication mutagenesis in Streptococcus pneumoniae: targeting fragment length is a critical parameter in use as a random insertion tool.

To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

Chimera

Isolation and characterization of three Streptococcus pneumoniae transformation-specific loci by use of a lacZ reporter insertion vector.

Although more than a dozen new proteins are produced when Streptococcus pneumoniae cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random lacZ transcriptional fusion library was constructed in S. pneumoniae by using the insertional lacZ reporter vector pEVP3. Screening the library for clones with competence-specific beta-galactosidase (beta-Gal) production yielded three insertion mutants with induced beta-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the lacZ reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of Bacillus subtilis com operons. One locus contained open reading frames (ORFs) homologous to the comEA and comEC genes in B. subtilis but lacked a comEB homolog. A second locus contained four ORFs with homology to the B. subtilis comG gene ORFs 1 to 4, but comG gene ORFs 5 to 7 were replaced in S. pneumoniae with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an erm cassette for gene disruptions.

Amino Acid Sequence

Sellar and suprasellar hemangiopericytoma mimicking pituitary adenoma.

Hemangiopericytoma is a vascular neoplasm of variable and unpredictable malignancy, composed of proliferating capillary pericytes surrounding endothelial-lined tubes or sprouts. The histological appearance is diagnostic. The treatment of primary intracranial hemangiopericytoma is surgical excision, supplemented by radiotherapy. Local tumor recurrence after many years is common, and late and widespread metastasis can occur. Long-term follow-up is mandatory.

Adenoma

Angioplasty and stenting of an unprotected left main bifurcation lesion.

A case of unprotected bifurcation left main disease treated on 2 occasions by angioplasty and stenting is presented. This case demonstrates the 2 main features of salvage angioplasty, namely medically refractory rest angina and refused bypass surgery. In addition, it presents short inflation time angioplasty for sole or main conduits and treating the left main as a bifurcation lesion.

Aged

Evolution of the genus Leishmania revealed by comparison of DNA and RNA polymerase gene sequences.

Previous hypotheses of Leishmania evolution are undermined by limitations in the phylogenetic reconstruction method employed or due to the omission of key parasites. In this experiment, sequences of the gene encoding the DNA polymerase alpha catalytic polypeptide (POLA) were analysed phylogenetically in combination with those encoding the RNA polymerase II largest subunit gene (RPOIILS) to infer a comprehensive phylogeny of Leishmania. Nineteen species of parasites were studied, comprising representatives of each Leishmania species-complex (Leishmania Leishmania tropica, Leishmania Leishmania donovani, Leishmania Leishmania mexicana, Leishmania Leishmania hertigi and Leishmania Viannia braziliensis), as well as parasites of questionable taxonomy (Leishmania herreri, Sauroleishmania adleri, Sauroleishmania deanei, Sauroleishmania gymnodactyli and Sauroleishmania tarentolae). The analyses presented here provide strong support for the hypothesis that the Leishmania that infect reptiles (also known as Sauroleishmania) evolved from mammalian Leishmania. One implication of this finding is that the taxonomic definition of Leishmania should be broadened to encompass characteristics of the reptilian parasites. However, this taxonomic revision is complicated in that Leishmania (L.) hertigi, Leishmania (L.) deanei and Leishmania herreri, which exhibit some biological properties of Leishmania, are more closely related to Endotrypanum on the basis of these sequence comparisons. Consequently, the taxonomic discrimination between Leishmania that infect mammals, Leishmania that infect reptiles and Endotrypanum may be more problematic than has been previously thought. Since our resulting phylogenetic hypothesis is supported by the analyses of two different genes, we speculate on the origin and evolutionary expansion of this lineage of kinetoplastid protozoa.

Animals

Coronary angioplasty for elderly patients with "high risk" unstable angina: short-term outcomes and long-term survival.

OBJECTIVES: We sought to compare the short- and long-term mortality rates in patients > or = 70 years old with unstable angina undergoing percutaneous transluminal coronary angioplasty (PTCA) with predicted coronary artery bypass graft surgery (CABG) short-term and U.S. census long-term mortality rates. BACKGROUND: Coronary angioplasty is an alternative revascularization strategy for patients with medically refractory rest angina and a high risk of adverse outcomes with CABG. Patients > or = 70 years old are a specific high risk subset. METHODS: A total of 131 consecutive patients aged > or = 70 years with unstable angina underwent PTCA; 82 (62%) of 131 had been refused CABG. Mortality over time was obtained from the Veterans Affairs Beneficiary Index Records Locator Subsystem. Predicted 30-day CABG-associated mortality was obtained from the Veterans Affairs Cardiac Risk Assessment Model. Mortality over time was expressed with Kaplan-Meier curves. RESULTS: The observed 30-day angioplasty survival rate was 87% compared with the predicted surgical 30-day survival rate of 85.5%. In those patients who survived 6 months after angioplasty (84%), their subsequent 1-, 2-, 3-, 4- and 5-year survival rates were comparable to age-matched subjects in the U.S. census. Mortality in certain subsets known to be at very high risk for CABG-for example, patients who had a previous CABG-was not high in this cohort of elderly subjects. The extremely high risk subsets identified in this PTCA cohort (shock, heart failure, pressors required, balloon pump required) were relatively infrequent subsets. CONCLUSIONS: For selected elderly patients with unstable angina deemed to be at "high risk" or even "prohibitive risk" for CABG, PTCA is an alternative revascularization strategy. The long-term mortality of successfully treated elderly patients is comparable to age-matched subjects. A prospective, multicenter, randomized trial of CABG versus PTCA, which includes patients > or = 70 years old, is being conducted (Veterans Affairs Cooperative Study 385: AWESOME).

Aged

Streptococcal competence for genetic transformation: regulation by peptide pheromones.

Although the capacity for genetic transformation is perhaps the most famous attribute of pneumococcus, use of this genetic phenomenon as a tool for study of the biology of the organism and of its pathogenicity has been largely restricted to a few favored unencapsulated strains, both by the delicacy of the conditions required for development of competence, and by experience that encapsulated strains transformed poorly. We discuss here the recent discovery of a small stable inexpensive peptide pheromone that acts as a quorum-sensing signal and that induces competence under a wide variety of conditions and in encapsulated strains. Its use circumvents some if not all limitations to the expression of transformability in pneumococcus and therefore expands opportunities for application of tools molecular genetics to many strains of pneumococcus without prior genetic manipulation.

Peptides