Pneumocystis pneumonia (PCP) remains a leading opportunistic infection in patients with weakened immune system. The fungus causing the infection belongs to the genus, Pneumocystis, and its members are found in a large variety of mammals. Although Pneumocystis carinii pneumonia is one of the leading causes of morbidity and mortality among patients with the acquired immunodeficiency syndrome, many questions about its epidemiology and transmission remain unanswered. Pneumocystis is classified as a fungus and is host-species specific, but an understanding of its reservoir, mode of transmission, and pathogenesis is incomplete. dihydropteroate synthase has been targeted in Pneumocystis pneumonia (PCP) disease DHPS is part of a trifunctional protein along with dihydroneopterin aldolase and hydroxyl methyl dihydro pterinpyrophospho kinase, two other enzymes in the folic acid biosynthesis pathway Sulfa resistance has evolved in numerous bacterial pathogens as well as in the malaria parasite, Plasmodium falciparum..In this present study, Structural characterization carried out the by various In silico tools. The predicted three dimensional structure depicts the structure based drug design.

Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.

Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100  U) or StuI (100 and 250  U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

ABSTRACT. Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α‐demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild‐type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid‐borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin‐layer chromatography, high‐performance liquid chromatography, and gas‐liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14‐desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild‐type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α‐methyl sterols better than the P. carinii enzyme.

Pneumocystis species are ascomycetous fungi that obligatorily dwell with no apparent ill effect in the lungs of normal mammals, but they become pathogenic when host defenses are compromised. Identified more than 100 years ago, these atypical fungi manifest characteristics that are unique within the Fungi, such as the lack of ergosterol, genetic complexity of surface antigens, and antigenic variation. Thought to be confined to the severely immunocompromised host, Pneumocystis spp. are being associated with new population niches owing to the advent of immunomodulatory therapies and increased numbers of patients suffering from chronic diseases. The inability to grow Pneumocystis spp. outside the mammalian lung has thwarted progress toward understanding their basic biology, but via the use of new genetic tools and other strategies, researchers are beginning to uncover their biological and genetic characteristics including a biphasic life cycle, significant metabolic capacities, and modulation of lifestyles.

Pneumocystis species are ascomycetous fungi that obligatorily dwell with no apparent ill effect in the lungs of normal mammals, but they become pathogenic when host defenses are compromised Identified more than 100 years ago, these atypical fungi manifest characteristics that are unique within the Fungi, such as the lack of ergosterol, genetic complexity of surface antigens, and antigenic variation Thought to be confined to the severely immunocompromised host, Pneumocystis spp are being associated with new population niches owing to the advent of immunomodulatory therapies and increased numbers of patients suffering from chronic diseases The inability to grow Pneumocystis spp outside the mammalian lung has thwarted progress toward understanding their basic biology, but via the use of new genetic tools and other strategies, researchers are beginning to uncover their biological and genetic characteristics including a biphasic life cycle, significant metabolic capacities, and modulation of lifestyles

Chromosomal rearrangements in human cancers are of two types, interchromosomal, which are rearrangements that involve exchange between loci located on different chromosomes, and intrachromosomal, which are rearrangements that involve loci located on the same chromosome. The type of rearrangement that typically activates a specific oncogene may be influenced by its nuclear location and that of its partner. In interphase nuclei, each chromosome occupies a distinct three-dimensional (3D) territory that tends to not overlap the territories of other chromosomes. It is also known that after double strand breaks in the genome, mobility of free DNA ends is limited. These considerations suggest that loci located deep within a chromosomal territory might not participate in interchromosomal rearrangements as readily as in intrachromosomal rearrangements. To test this hypothesis, we used fluorescence in situ hybridization with 3D high-resolution confocal microscopy to analyze the positions of six oncogenes known to be activated by recombination in human cancer cells. We found that loci involved in interchromosomal rearrangements were located closer to the periphery of chromosome territories as compared with the loci that were involved in intrachromosomal inversions. The results of this study provide evidence suggesting that nuclear architecture and location of specific genetic loci within chromosome territories may influence their participation in intrachromosomal or interchromosomal rearrangements in human thyroid cells.

The complex relationships between infectious organisms and their hosts often reflect the continuing struggle of the pathogen to proliferate and spread to new hosts, and the need of the infected individual to control and potentially eradicate the infecting population. This has led, in the case of mammals and the pathogens that infect them, to an 'arms race', in which the highly adapted mammalian immune system has evolved to control the proliferation of infectious organisms and the pathogens have developed correspondingly complex genetic systems to evade this immune response. We review how bacterial, protozoan and fungal pathogens from distant evolutionary lineages have evolved surprisingly similar mechanisms of antigenic variation to avoid eradication by the host immune system and can therefore maintain persistent infections and ensure their transmission to new hosts.

Spelling Pneumocystis jiroveciiTo the Editor: Our 2002 article in Emerging Infectious Diseases about nomenclature changes for organisms in the genus Pneumocystis (1) has been widely cited and probably will remain a source for persons seeking information about this subject.Therefore, we need to correct an error in 1 of the species names presented in our article and in the 1999 article by Frenkel (2) on which our article was based.In the 1999 article, Frenkel proposed that the species of Pneumocystis found in humans be named to honor the Czech parasitologist, Otto Jirovec.The 1999 article was his second proposal for this change.In 1976, he fi rst named the human pathogen Pneumocystis jiroveci (3), at which time it was classifi ed as a protozoan and therefore named according to the International Code of Zoological Nomenclature.By 1999, it had become clear that the organisms in the genus Pneumocystis are fungi, which are named according to the International Code of Botanical Nomenclature (ICBN) (4).Differences between the International Code of Zoological Nomenclature and ICBN resulted in the realization of an error in the species epithet proposed by Frenkel in 1999, and our 2002 article contained this error.Frenkel's 1999 article should have modifi ed the species epithet from "jiroveci" to "jirovecii," (ICBN Articles 32.7 and 60.11 and Rec.60C.1b).The correct and valid name under ICBN is Pneumocystis jirovecii.Redhead et al. further explain the basis for this correction (5).

BACKGROUND:The relationship between the parasitic fungus Pneumocystis carinii and its host, the laboratory rat, presumably involves features that allow the fungus to circumvent attacks by the immune system. It is hypothesized that the major surface glycoprotein (MSG) gene family endows Pneumocystis with the capacity to vary its surface. This gene family is comprised of approximately 80 genes, which each are approximately 3 kb long. Expression of the MSG gene family is regulated by a cis-dependent mechanism that involves a unique telomeric site in the genome called the expression site. Only the MSG gene adjacent to the expression site is represented by messenger RNA. Several P. carinii MSG genes have been sequenced, which showed that genes in the family can encode distinct isoforms of MSG. The vast majority of family members have not been characterized at the sequence level.RESULTS:The first 300 basepairs of MSG genes were subjected to analysis herein. Analysis of 581 MSG sequence reads from P. carinii genomic DNA yielded 281 different sequences. However, many of the sequence reads differed from others at only one site, a degree of variation consistent with that expected to be caused by error. Accounting for error reduced the number of truly distinct sequences observed to 158, roughly twice the number expected if the gene family contains 80 members. The size of the gene family was verified by PCR. The excess of distinct sequences appeared to be due to allelic variation. Discounting alleles, there were 73 different MSG genes observed. The 73 genes differed by 19% on average. Variable regions were rich in nucleotide differences that changed the encoded protein. The genes shared three regions in which at least 16 consecutive basepairs were invariant. There were numerous cases where two different genes were identical within a region that was variable among family members as a whole, suggesting recombination among family members.CONCLUSION:A set of sequences that represents most if not all of the members of the P. carinii MSG gene family was obtained. The protein-changing nature of the variation among these sequences suggests that the family has been shaped by selection for protein variation, which is consistent with the hypothesis that the MSG gene family functions to enhance phenotypic variation among the members of a population of P. carinii.

A recent article in Clinical and Experimental Dermatology by Saadat et al. described a purported case of botryomycosis caused by Pneumocystis carinii, which in turn prompted Mohapatra & Aggarwal to comment on the use of the name Pneumocystis carinii. Both publications contain incorrect information. Mohapatra & Aggarwal were critical of Saadat et al. for using the name Pneumocystis carinii for the human pathogen, Pneumocystis jirovecii. We concur with this opinion. However, we disagree with their opinion that there is no general consensus in the scientific community with regard to the nomenclature that should be used in reports about species in the genus Pneumocystis. P. carinii occurs in rats and has never been seen in humans, where the only species of Pneumocystis known is P. jirovecii (note the spelling of the species, it is not jiroveci). There is broad general agreement that human-associated Pneumocystis should be called P. jirovecii, a name that has been in use for nearly a decade and has been validated by the International Code of Botanical Nomenclature. However, not all authors agree with the consensus, as the letter from Mohapatra & Aggarwal illustrates. Nevertheless, Mohapatra & Aggarwal s reference to the Pneumocystis organism is a mischaracterization because there is no single organism named Pneumocystis. In addition, contrary to their statement, the species P. carinii was not renamed. It has been nearly a century since the name P. carinii was assigned to the species of Pneumocystis that is found in rats. This name has been retained; it is only the species infecting humans that has been named P. jirovecii. In the article by Saadat et al., the microbes observed were mischaracterized in several ways. In addition to referring to them as P. carinii, rather than P. jirovecii, the article indicates that the microbes in question are both protozoa and bacteria. Of course this is not possible. It should be noted that species in the genus Pneumocystis are neither protozoa nor bacteria, but true fungi. Additionally, the evidence that any species of Pneumocystis was involved in the botryomycosis formation is irreproducible and equivocal. The authors fail to indicate which of several commercially available monoclonal antibodies they used, hence the specificity cannot be gauged and the test cannot be reproduced. Additionally, it is difficult to discern the accentuated portions of the organism walls stained with Gomori methenamine silver (GMS) stain in the photomicrograph provided (Fig. 3), which they regard as a diagnostic characteristic. There are no magnification bars in this or the other micrographs, which also complicates interpretation. As GMS stains other fungi, the depicted morphology in that figure could be interpreted as arising from other fungi such as Lacazia loboi, which causes a keloidiform blastomycosis. These facts raise more questions, such as in which country was the patient examined, and where else in the world had he lived or travelled? None of this information was provided. Molecular sequence analysis could have been used to provide a definitive diagnosis of the organism. Until that is done, the report of Pneumocystis, under any species name, causing botryomycosis should be viewed with caution.

Journal Article Misinformation about Pneumocystis Get access R. Brubaker, R. Brubaker Department of Pathology, Christ Hospital, Cincinnati, OH, USA Search for other works by this author on: Oxford Academic Google Scholar S. A. Redhead, S. A. Redhead National Program on Environmental Health – Biodiversity, Agriculture and Agri‐Food Canada, Ottawa, Ontario, Canada Search for other works by this author on: Oxford Academic Google Scholar J. R. Stringer, J. R. Stringer Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for other works by this author on: Oxford Academic Google Scholar S. P. Keely, S. P. Keely Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for other works by this author on: Oxford Academic Google Scholar M. T. Cushion M. T. Cushion Research Service, Veterans Affairs Medical Center, Cincinnati, OHDepartment of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267‐0560, USA E‐mail: melanie.cushion@uc.edu Search for other works by this author on: Oxford Academic Google Scholar Clinical and Experimental Dermatology, Volume 34, Issue 7, 1 October 2009, Pages e426–e427, https://doi.org/10.1111/j.1365-2230.2009.03411.x Published: 01 October 2009 Article history Accepted: 20 December 2008 Published: 01 October 2009

To determine the relationship between aging, cell proliferation and mutation in different cell types, hearts, brains and kidneys from G11 PLAP mice between 1 week and 24 months of age were examined. Mutant cells were detected in tissue sections by staining for Placental Alkaline Phosphatase (PLAP) activity, an activity that marks cells that have sustained a frameshift mutation in a mononucleotide tract inserted into the coding region of the human gene encoding PLAP. The number of PLAP(+) cells increased with age in all three tissues. The types of cells exhibiting a mutant phenotype included cells that are proliferative, such as kidney epithelial cells, and cells that do not frequently replicate, such as cardiac muscle cells and neurons. In the brain, PLAP(+) cells appeared in various locations and occurred at similar frequencies in different regions. Within the cerebellum, PLAP(+) Purkinje cell neurons appeared at a rate similar to that seen in the brain as a whole. PLAP(+) cells were observed in kidney-specific cell types such as those in glomeruli and collecting tubules, as well as in connective tissue and blood vessels. In the heart, PLAP(+) cells appeared to be cardiac muscle cells. Regardless of tissue and cell type, PLAP(+) cells occurred as singletons and in clusters, both of which increased in frequency with age. These data show that age-associated accumulation of mutant cells occurs in diverse cell types and is due to both new mutation and proliferation of mutant cells, even in cell types that tend to not proliferate.

Loss of heterozygosity (LOH) in somatic cells can contribute to the genesis of cancer, but little is known about the frequency with which LOH occurs in normal cells of the body. To detect LOH in situ, we studied mouse shYFP embryonic stem (ES) cells and cells of the intestinal epithelia derived from these ES cells. shYFP ES cells are heterozygous at the ROSA26 locus. One copy of the locus carries a gene encoding a yellow fluorescent protein (YFP), while the other copy harbors an shRNA gene that produces a short hairpin RNA (shRNA) molecule that causes degradation of YFP mRNA. Nearly all cells in shYFP populations were faintly fluorescent, but brightly fluorescent cells arose at a rate of approximately 10(-5)bright cells/generation. Bright cells lacked the gene encoding the shRNA and contained two copies of the YFP gene. Comparison of these results to previous data on LOH in ES cells that lacked interfering shRNA showed that LOH in shYFP cells was not influenced by the presence of the shRNA. Bright cells were also seen in intestinal villi of chimeric mice made by injecting blastocysts with shYFP cells. These data demonstrate that this approach can detect LOH and suggest that it will allow detection of LOH in a broad array of tissues and cell types in transgenic mice made from shYFP cells.

ABSTRACT. Pneumocystis is a genus containing many species of non‐culturable fungi, each of which infects a different mammalian host. Pneumonia caused by Pneumocystis is a problem in immunodeficient humans, but not in normal humans. Nevertheless, it appears that Pneumocystis organisms cannot survive and proliferate outside of their mammalian hosts, suggesting that Pneumocystis parasitizes immunocompetent mammals. Residence in immunocompetent hosts may rely on camouflage perpetrated by antigenic variation. In P. carinii, which is found in rats, there exist three families of genes that appear to be designed to create antigenic variation. One gene family, which encodes the major surface glycoprotein (MSG), contains nearly 100 members. Expression of the MSG family is controlled by restricting transcription to the one gene that is linked to a unique expression site. Changes in the sequence of the MSG gene linked to the expression site occur and appear to be caused by recombination with MSG genes not at the expression site. Preliminary evidence suggests that gene conversion is the predominant recombination mechanism.

Analysis of the Pneumocystis murina MSG gene family and expression-site locus showed that, as in Pneumocystis carinii, P. murina MSG genes are arranged in head-to-tail tandem arrays located on multiple chromosomes, and that a variety of MSG genes can reside at the unique P. murina expression site. Located between the P. murina expression site and attached MSG gene is a block of 132 basepairs that is also present at the beginning of MSG genes that are not at the expression site. The center of this sequence block resembles the 28 basepair CRJE of P. carinii, but the block of conserved sequence in P. murina is nearly five times longer than in P. carinii, and much shorter than in P. wakefieldiae. These data indicate that the P. murina expression-site locus has a distinct structure.