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1.
BMC Mol Cell Biol ; 23(1): 17, 2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35399070

RESUMO

BACKGROUND: We have previously found that, in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5'-end were resistant to 5'-monophosphate requiring 5' → 3″ exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them. RESULTS: We now show the production of such 18S and 25S rRNAs in Saccharomyces cerevisiae that have been permanently switched to RNAP II (due to deletion of part of RNAP I upstream activator alone, or in combination with deletion of one component of RNAP I itself). They contain more than one phosphate at their 5'-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to have been modified in the cytoplasm. CONCLUSIONS: Our data confirm the existence of such molecules and firmly establish RNAP II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.


Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exonucleases/metabolismo , Fosfatos/metabolismo , RNA Polimerase II/metabolismo , RNA Ribossômico/genética , RNA Ribossômico 18S , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
BMC Mol Cell Biol ; 21(1): 59, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738873

RESUMO

BACKGROUND: We have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5' → 3' exonuclease, appearing as cells approached stationary growth phase. Initial analysis pointed to extra phosphate(s) at their 5'- end raising the possibility that they were newly transcribed. Here we report on additional experiments exploring this possibility and try to establish which of the RNA polymerases may be transcribing them. RESULTS: Oligo-ligation and primer extension again showed the presence of extra phosphate at the 5'-end of the reported processing sites for both 18S and 25S ribosomal RNA components. Inhibition of Pol I with BMH-21 increased the presence of the molecules. Quantitation with an Agilent Bioanalyzer showed that resistant 18S and 25S molecules are primarily produced in the nucleus. Utilizing an RNA cap specific antibody, a signal could be detected on these molecules via immunoblotting; such signal could be eliminated by decapping reaction. Both the cap specific antibody and eIF4E cap-binding protein, increased fold enrichment upon quantitative amplification. Antibodies specific for the RNA Polymerase II c-terminal domain and TFIIB initiator factor showed the presence of Pol II on DNA sequences for both 18S and 25S molecules in chromatin precipitation and qPCR assays. Rapamycin inhibition of TOR complex also resulted in an increase of resistant 18S and 25S molecules. CONCLUSIONS: These data raise the possibility of a role for RNA Polymerase II in the production of 18S and 25S molecules and indicate that efforts for more direct proof may be worthwhile. If definitively proven it will establish an additional role for RNA Polymerase II in ribosomal production.


Assuntos
Candida albicans/genética , Exonucleases/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Ribossômico 18S/genética , RNA Ribossômico/genética , Transcrição Gênica , Sequência de Bases , DNA Ribossômico/genética , Proteínas Fúngicas/metabolismo
3.
BMC Mol Biol ; 19(1): 1, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29351732

RESUMO

BACKGROUND: Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´â†’3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell. RESULTS: We have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5'-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior. CONCLUSIONS: Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.


Assuntos
Candida albicans/crescimento & desenvolvimento , Fosfodiesterase I/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Ribossômico 18S/metabolismo , RNA Ribossômico/metabolismo , Fosfatase Alcalina/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Pirofosfatases/metabolismo , RNA Fúngico/metabolismo , Sirolimo/farmacologia , Estresse Fisiológico , Serina-Treonina Quinases TOR/antagonistas & inibidores
4.
BMC Microbiol ; 17(1): 60, 2017 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-28284180

RESUMO

BACKGROUND: Candida krusei has been known to exhibit communal interactions such as pellicle formation and crawling out of nutritional broth. We noticed another possible interaction on agar surfaces, where C. krusei yeast cells formed mycelia along agar surfaces toward each other. We report here the results of experiments to study this interaction. RESULTS: When C.krusei yeast cells are plated in parallel streaks, they form mycelia along agar surfaces toward other yeasts. They also detect the presence of Candida albicans and Candida glabrata across agar surfaces, while the latter two react neither to their own kind, nor to C. krusei. Secreted molecule(s) are likely involved as C.krusei does not react to heat killed C. krusei. Timing and rate of mycelia formation across distances suggests that mycelia start forming when a secreted molecule(s) on agar surface reaches a certain concentration. We detected farnesol, tyrosol and tryptophol molecules that may be involved with mycelial formation, on the agar surfaces between yeast streaks. Unexpectedly the amounts detected between streaks were significantly higher than would have expected from additive amounts of two streaks. All three Candida species secreted these molecules. When tested on agar surface however, none of these molecules individually or combined induced mycelia formation by C. krusei. CONCLUSIONS: Our data confirms another communal interaction by C. krusei, manifested by formation of mycelia by yeast cells toward their own kind and other yeasts on agar surfaces. We detected secretion of farnesol, tyrosol and tryptophol by C. krusei but none of these molecules induced this activity on agar surface making it unlikely that they are the ones utilized by this yeast for this activity.


Assuntos
Ágar/química , Candida/crescimento & desenvolvimento , Candida/fisiologia , Interações Microbianas/fisiologia , Micélio/crescimento & desenvolvimento , Candida/efeitos dos fármacos , Candida/metabolismo , Candida albicans/metabolismo , Candida albicans/fisiologia , Candida glabrata/metabolismo , Candida glabrata/fisiologia , Meios de Cultura , Farneseno Álcool/farmacologia , Indóis/farmacologia , Micélio/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologia
5.
PLoS One ; 10(5): e0124430, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25946110

RESUMO

Candida albicans is an important polymorphic human pathogen. It can switch from a unicellular yeast form to germinating hypha, which may play a role in making it the successful pathogen it is. This hyphal transformation can be triggered by various extracellular stimuli, the most potent one being serum from any source. We have previously reported that Candida albicans transiently polyadenylates portions of both the large and small subunits of ribosomal RNA, shortly after serum exposure. Northern blots at the same time suggested that serum might induce a decrease in total ribosomal RNA. We have carried out a number of experiments to carefully assess this possibility and now report that serum significantly reduces ribosomal RNA in Candida albicans. Fluorometric measurements, Northern blotting and quantitative RT-PCR, have all confirmed this decrease. Timed experiments show that serum induces this decrease rapidly, as it was seen in as early as five minutes. Cell mass is not decreased as total cellular protein content remains the same and metabolic activity does not appear to slow, as assessed by XTT assay, and by the observation that cells form hyphal structures robustly. Another hyphal inducer, N-acetylglucosamine, also caused RNA decrease, but to a lesser extent. We also observed it in non-germinating yeast, such as Candida glabrata. The reason for this decrease is unknown and overall our data suggests that decrease in rRNA does not play a causal role in hyphal transformation. Rapid and significant decrease in a molecule so central to the yeast's biology is of some importance, and further studies, such as its effect on protein metabolism, will be required to better understand its purpose.


Assuntos
Candida albicans/efeitos dos fármacos , RNA Ribossômico/genética , Soro/microbiologia , Acetilglucosamina/farmacologia , Animais , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Bovinos , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento
6.
Am J Manag Care ; 19(9): e317-24, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24449959

RESUMO

OBJECTIVES: To evaluate our outpatient parenteral antimicrobial therapy (OPAT) program to determine its impact on infection management in a facility notable for high patient comorbidity and a large catchment area that includes most of Southern California. STUDY DESIGN: Retrospective chart review. METHODS: We reviewed all episodes of patients receiving OPAT from our institution from 2006 through 2009 for patient utilization characteristics and assessment of complications. RESULTS: A total of 333 patients received 393 courses of OPAT for a mean of 21.1 days. Diabetes mellitus (53.5%), psychiatric disease (39%), and chronic kidney disease (31%) were common; more than half the patients lived more than 20 miles from our medical center. Osteomyelitis (39.7%) and bacteremia (19.3%) accounted for the majority of OPAT indications. Staphylococcus aureus (36.4%) was the most frequent infecting organism, and vancomycin (37.4%) was the most frequently prescribed medication. Complications including hospital readmission, adverse drug reactions, or line-related complications were noted in 96 of 393 (24.4%) episodes, but most were minor, reversible, or not directly related to the OPAT given. Serious line-related complications that required hospital readmission were noted in only 6 (1.5%) episodes. OPAT was completed as planned in 313 (79.6%) episodes; end-stage renal disease was associated with OPAT noncompletion in multivariable analysis (odds ratio = 2.20, P = .047). We estimated that OPAT saved our medical center $4 million per year. CONCLUSIONS: Despite our patients' high level of comorbidity and our facility's large catchment area, we were able to deliver OPAT successfully and safely with significant cost savings.


Assuntos
Assistência Ambulatorial , Anti-Infecciosos/administração & dosagem , Hospitais de Veteranos , California , Feminino , Humanos , Infusões Parenterais , Masculino , Auditoria Médica , Adesão à Medicação , Pessoa de Meia-Idade , Ambulatório Hospitalar , Readmissão do Paciente , Estudos Retrospectivos
7.
BMC Res Notes ; 4: 74, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21426556

RESUMO

BACKGROUND: Candida species including Candida krusei have become common pathogens, especially in immune-compromised patients. Pellicle on the surface of incubating nutrient broth extending with an adherent film above the broth has been described as a feature of this organism. We investigated whether this easily observable adherent film could be useful in the identification of this yeast. We also wanted to see if this process involved any morphological changes from the yeast form on the part of C. krusei. FINDINGS: Common and less frequently isolated species of Candida were inoculated into YPD broth and observed for pellicle formation. For C. krusei different inoculum sizes and time periods were studied to establish earliest period and the smallest number of organisms needed for this process. A cover-slip assay was established to observe the architecture of the film formed by this organism. Among the clinically common Candida species, only C. krusei formed a visible film, requiring 106 organisms to produce it at 24 hours post inoculation. Film formation also differentiated C. krusei from C. inconspicua usually reported as a complex by carbohydrate assimilation assays. Rarely isolated C. famata and C. norvegensis also formed pellicles and film but less robustly. Microscopic observations of the film showed only yeast forms, no hypha or pseudohypha were seen. CONCLUSIONS: Pellicle formation following inoculation of a clinical specimen into liquid culture, is a useful alert to the probable presence of C. krusei and likely fluconazole resistance, while awaiting the results of more definitive identification assays. Pellicle and adherence film formation are not a result of polymorphic changes on the part of C. krusei as only yeast forms were present.

8.
BMC Mol Biol ; 5: 17, 2004 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-15461824

RESUMO

BACKGROUND: Candida albicans is a polymorphic fungus causing serious infections in immunocompromised patients. It is capable of shifting from yeast to germinating forms such as hypha and pseudohypha in response to a variety of signals, including mammalian serum. We have previously shown that some of the large 25S components of ribosomal RNA in Candida albicans get polyadenylated, and this process is transiently intensified shortly after serum exposure just prior to the appearance of germination changes. RESULTS: We now present data that this process also involves the small 18S subunit of ribosomal RNA in this organism. Unlike the large 25S subunit, polyadenylation sites near the 3' end are more variable and no polyadenylation was found at the reported maturation site of 18S. Similar to 25S, one or more polyadenylated mature sized 18S molecules get intensified transiently by serum just prior to the appearance of hypha. CONCLUSIONS: The transient increase in polyadenylation of both the large and the small subunits of ribosomal RNA just prior to the appearance of hypha, raises the possibility of a role in this process.


Assuntos
Candida albicans/genética , Poliadenilação/genética , RNA Ribossômico 18S/genética , RNA Ribossômico/genética , Clonagem Molecular/métodos , Poli A/sangue , Poli A/genética , RNA Fúngico/sangue , RNA Fúngico/genética , RNA Ribossômico 18S/sangue
9.
Artigo em Inglês | MEDLINE | ID: mdl-12931094

RESUMO

OBJECTIVES: The purpose of this study was to develop a reproducible, quantitative model of Candida albicans adhesion to human dentin through the use of a colorimetric method and to evaluate the effect of smear layer on candidal adhesion. STUDY DESIGN: Dentin disks with or without smear layer were incubated with C albicans (10(8) cells/mL) for 4 hours. After incubation, the disks were exposed to an (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide-coenzyme Q solution for 2 hours. The color of (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide formazan in the supernatant was determined spectrophotometrically at 492 nm. To relate formazan formation to cell numbers, standard curves were generated with known numbers of yeast cells without dentin. The number of adherent cells per square millimeter was then calculated. RESULTS: The number of attached C albicans cells was 2.4 x 10(4) per square millimeter in dentin with smear layer and 1.5 x 10(4) in dentin without smear layer (P <.05). CONCLUSION: (2,3)-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide assay is a potential microbiologic tool for the quantitative determination of Candida adhesion to human dentin.


Assuntos
Candida albicans/fisiologia , Dentina/microbiologia , Camada de Esfregaço , Adesão Celular/fisiologia , Contagem de Colônia Microbiana , Colorimetria , Formazans , Humanos , Indicadores e Reagentes , Espectrofotometria , Sais de Tetrazólio , Fatores de Tempo , Ubiquinona
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