No abstract

For 50 years, physiologic studies in Candida albicans have associated fermentation with filamentation and respiration with yeast morphology. Analysis of the mitochondrial proteome of a C. albicans NDH51 mutant, known to be defective in filamentation, identified increased expression of several proteins in the respiratory pathway. Most notable was a 15-fold increase in pyruvate dehydrogenase complex protein X (Pdx1), an essential component of the pyruvate dehydrogenase complex. In basal salts medium with < or = 100 mM glucose as carbon source, two independent pdx1 mutants displayed a filamentation defect identical to ndh51; reintegration of one PDX1 allele restored filamentation. Concentrations of glucose < or = 100 mM did not correct the filamentation defect. Expanding on previous work, these studies suggest that increased expression of proteins extraneous to the electron transport chain compensates for defects in the respiratory pathway to maintain yeast morphology. Mitochondrial proteomics can aid in the identification of C. albicans genes not previously implicated in filamentation.

The chromosomal location of the human gene for erythropoietin (EPO) was determined by Southern blot hybridization analysis of a panel of human-mouse somatic hybrid cell DNAs. DNAs from cell hybrids containing reduced numbers of human chromosomes were treated with the restriction enzyme PstIand screened with a cloned human EPO cDNA probe. EPO is assigned to human chromosome 7 based on the complete cosegregation of EPO with this chromosome in all 45 cell hybrids tested. A cell hybrid containing a translocated derivative of chromosome 7 localizes EPO to 7pter→q22. A HindIII restriction fragment length polymorphism is detected by hybridization of the EPO cDNA probe to human genomic DNA.

No abstract

By inhibiting the conversion of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) to mevalonate, statins impair cholesterol metabolism in humans. We reasoned that statins might similarly interfere with the biosynthesis of ergosterol, the major sterol of the yeast cell membrane. As assessed by spectrophotometric and microscopic analysis, significant inhibition of biofilm production was noted after 16-h incubation with 1, 2.5, and 5 M simvastatin, concentrations that did not affect growth, adhesion, or hyphal formation by C. albicans in vitro. Higher concentrations (10, 20, and 25 M simvastatin) inhibited biofilm by Ͼ90% but also impaired growth. Addition of exogenous ergosterol (90 M) overcame the effects of 1 and 2.5 M simvastatin, suggesting that at least one mechanism of inhibition is interference with ergosterol biosynthesis. Clinical isolates from blood, skin, and mucosal surfaces produced biofilms; biofilms from bloodstream isolates were similarly inhibited by simvastatin. In the absence of fungicidal activity, simvastatin's interruption of a critical step in an essential metabolic pathway, highly conserved from yeast to man, has unexpected effects on biofilm production by a eukaryotic pathogen. (Pediatr Res 66: 600-604, 2009)

A human genomic DNA fragment in phage lambda containing FSHB, the gene for the beta-subunit of human follicle-stimulating hormone (FSH-beta), was analyzed and the nucleotide sequence of the region of the clone encoding FSH-beta was determined. A subclone of the lambda phage containing 67% of FSH-beta coding sequence was used as hybridization probe to determine the human chromosomal location of FSHB. A panel of mouse-human somatic cell hybrids containing reduced numbers of human chromosomes was screened with the FSHB probe; complete cosegregation of FSHB with human chromosome 11 was observed in all 26 cell hybrids tested. Analysis of a set of cell hybrids containing translocated derivatives of chromosome 11 further localized FSHB to the human chromosome region 11p11.2----11pter. A Hind III restriction fragment length polymorphism (RFLP) detected by another subclone of the lambda phage containing FSHB now provides a genetic marker for this region of the human genome.

In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-β (TGF-β) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-β control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-β signaling pathway by combining measurements of the levels and the activation state of Smad signaling intermediates with DNA-based diagnostic assays. In this report, we demonstrate the validity of this approach using a panel of seven squamous cell carcinoma (SCC) lines known to be refractory to TGF-β-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and -3. Furthermore, treatment with TGF-β induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-β type II receptor. Three of the remaining SCC lines failed to express the common mediator Smad4, two on the basis of loss of transcription and one by a posttranscriptional mechanism. Thus, a mechanism for TGF-β resistance was identified in five of the seven tumor cell lines. Interestingly, in the two remaining lines, no abnormalities of signaling intermediates were found, and TGF-β was able to activate TGF-β-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-β is due to factors extraneous to the TGF-β pathway itself. Application of our protein-based strategy to interrogate the TGF-β signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-β resistant in vivo.

High SVR rates were achieved in patients infected with GT2 treated with daclatasvir-based regimens irrespective of GT2 subtype or baseline NS5A polymorphisms.