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M T Cushion

Publications and source records attributed to M T Cushion.

At least 37 records · Page 2Linked to original sources
PubMed1998

The genome of Pneumocystis carinii.

The best understood special form of P. carinii, P. carinii formae specialis (f.sp.) carinii, appears to be haploid and contains about 8 million base pairs of DNA (8.5 fg) per nucleus. The genome of P. carinii f.sp. carinii is divided into 13-15 linear chromosomes that range from 300 to 700 kb in size. Eight different P. carinii f.sp. carinii karyotypes have been observed. The karyotypes of P. carinii f.sp. carinii differ due to slight variations in the lengths of chromosomes, but the 8 karyotype-forms of P. carinii f.sp. carinii exhibit very little variation in DNA sequence. By contrast, the genome of P. carinii f.sp. carinii differs markedly in sequence from the genomes of P. carinii from other hosts, such as mouse, ferret and human. In addition, chromosomes and DNA sequences from P. carinii from mouse, ferret, and human also differ greatly from each other. The genome of a ferret P. carinii appears to be up to 1.7 times larger than those of P. carinii from other hosts. Nearly two dozen P. carinii genes have been cloned and sequenced. The typical P. carinii gene sequence is 60-65% A+T. P. carinii genes usually contain introns, which are typically less than 50 bp in length, but can be as numerous as 9 per gene. A system for naming P. carinii genes is proposed in which each gene would be designated by an italic three-letter lower case symbol. The first allele (i.e. sequence) that is found would have a superscript 1, such as xyz1(1). Any subsequent alleles would be designated as xyz1(2), etc. A protein would have the same symbol as the gene that produced it, but written in roman print with the first letter an uppercase, such as Msg1. Some of the P. carinii genome is comprised of DNA sequences that are present dozens of times. Three families of such repeated DNA sequences have been described. Two of these families (MSG and PRT) encode proteins. The third family is the telomere repeat, which is found at the ends of each chromosome, and sometimes at internal chromosomal sites, in which case it has been called the alpha repeat. Determination of the complete sequence of the P. carinii genome is both practicable and of primary importance to the understanding of this organism. The small size of the P. carinii genome and its packaging into chromosomes that are resolvable by PFGE will facilitate sequence analysis.

Chromosomes, Fungal↗
PubMed1998

Taxonomy, genetic organization, and life cycle of Pneumocystis carinii.

Pneumocystis carinii was initially misidentified as a protozoan parasite. Recent molecular and biochemical analyses provide unequivocal evidence for placement of P. carinii with the fungi, and that P. carinii is most likely an ascomycete. Genetic investigations further show that P. carinii derived from different mammalian hosts (human, rat, mouse, and ferret) exhibit considerable chromosomal and gene sequence divergence indicating that they are likely of different species. The life cycle of P. carinii has not been definitively established, but available evidence is reviewed in light of classification of this organism as a fungus.

Animals↗
PubMed1997

Effects of steroidal allenic phosphonic acid derivatives on the parasitic protists Leishmania donovani, Leishmania mexicana mexicana, and Pneumocystis carinii carinii.

Several pathogenic fungi and protozoa are known to have sterols distinct from those of their mammalian hosts. Of particular interest as targets for drug development are the biosyntheses of the sterols of important parasites such as the kinetoplastid flagellates and the AIDS-associated opportunistic protist Pneumocystis carinii. These pathogens synthesize sterols with an alkyl group at C-24, and some have a double bond at C-22 of the side chain. Humans and other mammalian hosts are incapable of C-24 alkylation and C-22 desaturation. In the present study, three steroidal compounds with side chains substituted by phosphonyl-linked groups were synthesized and tested for their effects on Leishmania donovani and L. mexicana mexicana culture growth. The compounds inhibited organism proliferation at concentrations in micrograms per milliliter. The most potent inhibitors of this group of compounds were characterized by two ethyl groups at the phosphate function. Leishmania organisms treated with 17-[2-(diethylphosphonato) ethylidienyl]3-methoxy-19-norpregna-1,3,5-triene exhibited reduced growth after transfer into inhibitor-free medium. Because there are currently no axenic methods available for the continuous subcultivation of P. carinii, the effects of these drugs on this organism were evaluated by two alternative screening methods. The same two diethyl phosphonosteroid compounds that inhibited Leishmania proliferation were also the most active against P. carinii as determined by the potent effect they had on reducing cellular ATP content. Cystic as well as trophic forms responded to the drug treatments, as evaluated by a dual fluorescent staining live-dead assay. Other modifications of steroidal phosphonates may lead to the development of related drugs with increased activity and specificity for the pathogens.

Adenosine Triphosphate↗
PubMed1997

A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii agents.

A series of over 60 agents representing several different classes of compounds were evaluated for their effects on the ATP pools of Pneumocystis carinii populations derived from immunosuppressed rats. A cytotoxicity assay based on an ATP-driven bioluminescent reaction was used to determine the concentration of agent which decreased the P. carinii ATP pools by 50% versus untreated controls (IC50). A ranking system based on the IC50 value was devised for comparison of relative responses among the compounds evaluated in the cytotoxic assay and for comparison to in vivo efficacy. With few exceptions, there was a strong correlation between results from the ATP assay and the performance of the compound in vivo. Antibiotics, with the exception of trimethoprim-sulfamethoxazole (TMP-SMX), were ineffective at reducing the ATP pools and were not active clinically or in the rat model of P. carinii pneumonia. Likewise, other agents not expected to be effective, e.g., antiviral compounds, did not show activity. Standard anti-P. carinii compounds, e.g., TMP-SMX, pentamidine, and dapsone, dramatically reduced ATP levels. Analogs of the quinone and topoisomerase inhibitor groups were shown to reduce ATP concentrations and hold promise for further in vivo investigation. The cytotoxicity assay provides a rapid assessment of response, does not rely on replicating organisms, and should be useful for assessment of structure-function relationships.

Adenosine Triphosphate↗
PubMed1997

Molecular epidemiology and antibiotic susceptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey.

To determine patterns of antimicrobial susceptibility among enterococci and to assess molecular characteristics of vancomycin-resistant enterococci, 157 clinical blood isolates of enterococci from 10 hospitals in Cincinnati, Ohio, were prospectively collected during a 6-month period from February to July 1995. The isolates included 108 (69%) E. faecalis isolates, 46 (29%) E. faecium isolates, and 1 isolate each of E. avium, E. durans, and E. gallinarum. The E. faecalis and E. faecium isolates differed in their susceptibilities to ampicillin (100 versus 20%), ampicillin-sulbactam (100 versus 13%), vancomycin (100 versus 57%), imipenem (94 versus 2%), and high levels of gentamicin (59 versus 83%). Supplemental susceptibility testing of the 21 vancomycin-resistant isolates showed that 21 (100%) were susceptible to chloramphenicol and that only 7 (33%) were susceptible to doxycycline. Nineteen (90%) of the vancomycin-resistant E. faecium isolates were of the VanB phenotype, with vanB resistance genes detected by PCR and hybridization with gene-specific probes; and the E. gallinarum isolates demonstrated the VanC phenotype with the vanC1 gene. One vancomycin-resistant E. faecium isolate was highly resistant to both teicoplanin and vancomycin, corresponding to the VanA phenotype; however, it was found to have the vanB gene. Pulsed-field gel electrophoresis (PFGE) revealed that all of the 19 E. faecium isolates with the VanB phenotype had identical to closely related banding patterns. Hybridization of restriction enzyme-digested DNA separated by PFGE with a vanB gene probe demonstrated differences in the locations of vanB genes that corresponded closely to the PFGE banding patterns. Our study has documented that the emerging vancomycin resistance in our city was mainly due to the clonal dissemination of a single strain of E. faecium VanB.

Anti-Bacterial Agents↗
PubMed1996

Antigenic differences associated with genetically distinct Pneumocystis carinii from rats.

Pneumocystis carinii is a family of organisms found in a wide variety of mammalian lungs. In immunocompromised hosts, the organisms are able to produce an oftentimes fatal pneumonia. The existence of distinct types of Pneumocystis populations is strongly supported by antigenic and genetic evidence. In the present study, we assessed the antigenic profiles of two genetically distinct Pneumocystis carinii populations, P. carinii f. sp. carinii and P. carinii f. sp. ratti, as well as two types of P. carinii f. sp. carinii defined by electrophoretic karyotyping (forms 1 and 2). The separated and blotted proteins of the organism preparations were probed with four monoclonal antibodies (MAbs) generated to the major surface glycoproteins of rat-derived P. carinii, one anti-human P. carinii MAb, and two polyclonal antisera made with rat-derived P. carinii as the immunogen. Differences in reactivities between the P. carinii f. sp. carinii and P. carinii f. sp. ratti preparations were detected with two of the MAbs, and both of the rat P. carinii polyclonal antisera in the 45- to 55-kDa molecular mass range, but not with the human P. carinii MAb. The reactivities of the 16 P. carinii f. sp. carinii preparations were the same with two exceptions. Two preparations of form 1 showed strong reactivity with the anti-MSG MAb RA-C11. The ratios of cyst forms to trophic forms evaluated by microscopy were not associated with any of the differences observed in the antigenic profiles. The antigenic differences between P. carinii f. sp. carinii and P. carinii f. sp. ratti are consistent with the distinction of these two populations made by molecular genetic techniques, while the two differences detected among the P. carinii f. sp. carinii preparations suggest the organism may be able to modulate antigenic epitopes. The use of immunoblotting to differentiate infecting organism populations and assess antigenic modulation holds promise for future epidemiologic studies.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Use of semiquantitative PCR to assess onset and treatment of Pneumocystis carinii infection in rat model.

The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.

Animals↗
PubMed1994

Fractionation of Pneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations.

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.

Animals↗
PubMed1994

Analysis of Pneumocystis carinii organism burden, viability and antigens in bronchoalveolar lavage fluid in AIDS patients with pneumocystosis: correlation with disease severity.

OBJECTIVES: We examined 96 bronchoalveolar lavage fluid (BALF) specimens from AIDS patients with proven Pneumocystis carinii pneumonia (PCP) in order to compare the relationship of organism burden, viability and antigen expression with disease severity at the time of clinical presentation. METHODS: Tinctorial analysis of BALF specimens with proven PCP using Diff-Quik, cresyl echt violet and erythrosin B stains to evaluate organism burden and viability. P. carinii antigen examination was performed by Western blot analysis. RESULTS: P. carinii cluster ratios were more sensitive than cyst counts as an indicator of organism burden, and correlated well with the alveolar-arterial oxygen gradient as a measure of disease severity. Erythrosin B, the vital stain used to measure P. carinii viability, displayed a wide range of values and provided little useful information. Antigens of 35-45 and 95kD, which were specific for P. carinii, were found by immunoblot analysis in BALF cellular fraction of most patients with pneumocystosis. By contrast, antigens of 52 and 66 kD, which were found in both BALF supernatant and cellular fractions of P. carinii patients and controls, most likely represented albumin and immunoglobulin G heavy chain, respectively, of host origin. The 35-45 kD antigen was found in 88% of the BALF specimens and appeared to represent an important marker of P. carinii infection. The 95 kD antigen was detected in 49% of the specimens. CONCLUSIONS: We conclude that analysis of P. carinii characteristics in BALF specimens of patients with pneumocystis may provide additional information. These data will also be helpful in developing more sensitive assays and in targeting specific P. carinii factors for future investigation.

AIDS-Related Opportunistic Infections↗
PubMed1994

Use of an ATP bioluminescent assay to evaluate viability of Pneumocystis carinii from rats.

A bioluminescent assay which employs the luciferin-luciferase ATP-dependent reaction was used to evaluate the viability of populations of Pneumocystis carinii derived from infected rat lungs. Contamination with host cells was reduced by a purification method which involved a combination of low- and high-speed centrifugations resulting in a 1,000-fold reduction of the rat cells while enriching for the trophic form of P. carinii. A linear correlation for the number of P. carinii nuclei versus the amount of ATP was observed. The ATP content of the organism populations could be maintained at inoculum levels for one week, although the number of organisms did not increase. Addition of respiratory chain inhibitors dramatically decreased the ATP content of the P. carinii after 24 h of incubation, with the exception of the antibiotic oligomycin B. Low concentrations of trimethoprim-sulfamethoxazole and pentamidine isethionate reduced the organism ATP content by over 50% after 24 h of exposure, while no effect was observed with 100-fold greater concentrations of ampicillin. The bioluminescent assay was found to be a more sensitive indicator of viability than a dual fluorescent staining technique. This assay does not require replication of P. carinii and should be a useful method for in vitro drug screening and viability assessment of P. carinii populations.

Adenosine Triphosphate↗
PubMed1993

Genetic stability and diversity of Pneumocystis carinii infecting rat colonies.

There is increasing molecular and antigenic evidence that Pneumocystis carinii organisms isolated from humans, ferrets, and rats are different species. In contrast, little is known about the extent of genetic diversity among P. carinii strains found within a single mammalian species. In the present study, electrophoretic karyotypes were obtained from P. carinii prepared from 10 chronically immunosuppressed rat colonies to investigate diversity at the chromosomal level. Most organism preparations produced patterns with 13 to 15 bands, but as many as 24 bands were observed in a few preparations. All bands separated between 700 and 300 kbp. Four distinct karyotype forms emerged from among the 13- to 15-band karyotypes of the 10 colonies sampled. Form 1 was shared by five rat strains from two vendors; form 2 was shared by two rat strains from the same vendor; and forms 3 and 4 were unique to their vendor colonies. Within a given rat colony, most rats harbored the same P. carinii karyotype. A survey of selected rat colonies showed that the karyotype within a vendor colony could remain stable over a period of 2 to 3 years. Hybridization of the blotted karyotypes with a repetitive DNA element isolated from rat-derived P. carinii and with single-copy gene probes showed that every chromosome in the karyotypes contained some repetitive DNA, and there was a general size concordance among the chromosomes carrying the unique gene loci. Differences in gene sequences, electrophoretic karyotypes, and hybridization profiles suggested that the immunosuppressed rats were infected by genetically distinct P. carinii strains. A provisional system of nomenclature for P. carinii that will permit differentiation of P. carinii organisms from the same mammalian host is discussed. These data show that all rats were not infected by a single type of P. carinii, that pulsed-field gradient electrophoresis can detect sufficient genetic diversity among the organism preparations to allow for characterization of the organisms, and that the genome of the organism within the rat host is relatively stable over time.

Animals↗
PubMed1993

Molecular characterization of a novel repetitive element from Pneumocystis carinii from rats.

A repetitive DNA sequence, Rp-alpha, was isolated from rat-derived Pneumocystis carinii. The genome of rat-derived P. carinii contained 10 to 15 copies of Rp-alpha, which were located on most chromosomes, but no Rp-alpha could be detected in P. carinii derived from either humans or mice. Sequence analysis of two copies of the repeat showed them to be related but distinct. Each of them contained several copies of the 9-base sequence TAACCCTAA, arranged as direct repeats. Oligonucleotides consisting of multimers of this 9mer hybridized to the same set of chromosomes recognized by cloned copies of the Rp-alpha repeat. When used in DNA fingerprinting, the Rp-alpha repeat was capable of distinguishing between different isolates of rat-derived P. carinii.

Animals↗
PubMed1993

Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats.

Pneumocystis carinii pneumonia is an oftentimes fatal infection for hosts in an immunocompromised state. The disease occurs in a wide variety of mammals, but the etiologic agent of this disease has been referred to as P. carinii regardless of the host species. However, even within a single host species, such as laboratory rats, distinct varieties of P. carinii have been identified from differences in the electrophoretic migration of chromosomes in agarose gels. Here we present evidence indicating that some laboratory rats can contain two different genetic variants of P. carinii that differ not only in electrophoretic karyotype but also in the presence of a particular repeated DNA sequence, in the presence of an intron in the 18S ribosomal RNA gene, and in the sequence of part of the 18S rRNA gene. Most of the rat colonies studied were infected with P. carinii that contained the repeated DNA and the 18S rRNA gene intron. The other type of rat-derived P. carinii, which lacked the repeated DNA and the intron in the 18S rRNA gene, was found as a coinfection with the first. Parasite populations from different coinfected rats contained the two variants in different proportions.

Animals↗
PubMed1992

Flow cytometric analyses of lectin binding to Pneumocystis carinii surface carbohydrates.

Pneumocystis carinii obtained from infected rat lung homogenates was incubated with fluorescein isothiocyanate-conjugated lectins, counterstained with the nuclear stain, propidium iodide (PI), and analyzed by dual parameter histograms for lectin-associated green and PI-associated red fluorescence using a fluorescence-activated cell sorter. The presence of glucose/mannose moieties was evidenced by the binding of all organisms to concanavalin A and Wisteria floribunda. From the lectin group specific for N-acetyl-D-glucosamine, P. carinii reacted strongly with wheat germ agglutinin and less intensely with Solanum tuberosum. Reaction with lectins specific for N-acetyl-D-galactosamine/galactose was variable, probably reflecting the secondary binding affinities of the lectins used. Soybean agglutinin, Bauhinia purpurea agglutinin, and Maclura pomifera agglutinin reacted moderately, whereas Dolichos biflorus agglutinin, and Griffonia simplicifolia I reacted less avidly. The organisms reacted partially with Ulex europaeus agglutinin, a lectin specific for fucose, and did not react well with Arachis hypogaea, Viscum album agglutinin, and Griffonia simplicifolia I beta 4, lectins specific for galactose. A very weak fluorescent signal was detected with Limax flavus agglutinin, suggesting little or no sialic acid was present. All lectin-binding reactions were confirmed for specificity by inhibition with the relevant carbohydrates. Flow cytometric analysis of lung-derived Pneumocystis organisms stained with fluorescent surface and nuclear dyes provides a rapid method for characterization of large parasite populations.

Animals↗