The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.

Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.

The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.

Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products.

An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth.

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Delta cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.

Ethnomedicine and dominant medicine in multicultural Australia: a critical realist reflection on the case of Korean-Australian immigrants in Sydney.

BACKGROUND: Viewed through the micro focus of an interpretive lens, medical anthropology remains mystified because interpretivist explanations seriously downplay the given context in which individual health seeking-behaviours occur. This paper draws upon both the interpretivist and political economy perspectives to reflect on the ethno medical practices within the Korean-Australian community in Sydney. METHODS: We draw on research data collected between 1995 and 1997 for an earlier study of the use of biomedical and traditional medicine by Korean-Australians in Sydney. A total of 120 interviews were conducted with a range of participants, including biomedical doctors, traditional health professionals, Korean community leaders and Korean migrants representing a range of socio-economic backgrounds and migration patterns. RESULTS AND DISCUSSION: First, the paper highlights the extent to which the social location of migrants in a host society alters or restructures their initial cultural practices they bring with them. Second, taking hanbang medicine in the Korean-Australian community as an illustrative case, the paper explores the transformation of the dominant biomedicine in Australia as a result of the influx of ethnomedicine in the era of global capitalism and global movement. CONCLUSION: In seeking to explain the popularity and supply of alternative health care, it is important to go beyond the culture of each kind of health care itself and to take into consideration the changes occurring at societal, national and global levels as well as consequential individual response to the changes. New social conditions influence the choice of health care methods, including herbal/alternative medicine, health foods and what are often called New Age therapies.

Sialoendoscopic treatment for radioiodine induced sialadenitis.

BACKGROUND AND OBJECTIVE: The use of radioiodine (RI) for the ablation of residual thyroid tissue and metastatic thyroid cancer lesions after thyroidectomy has become established as standard treatment in the management of differentiated thyroid cancer and subsequent sialadenitis is the most common complication of RI therapy. The purpose of this study was to establish a new treatment modality for RI-induced sialadenitis. METHOD: The study group consisted of 115 patients with a mean age of 47.7 (range, 24-78) years. All patients received RI therapy after total thyroidectomy. The incidence of RI-induced sialadenitis, salivary gland involvement, administered RI dose, treatment modality, and result of treatment by interventional sialoendoscopy were evaluated. RESULTS: The incidence of RI-induced sialadenitis was 18% (21/115), with involvement of the parotid more frequent than the submandibular gland. The average development period of RI-induced sialadenitis was 4.8 months. The average RI dosage for the sialadenitis group was higher than for the nonsialadenitis group, suggesting that RI-induced sialadenitis may be dose related, although the data were not statistically significant because of the small numbers in the high-dose group. Conservative management was effective in 71% (15/21) of the cases, and interventional sialoendoscopy was successful in 50% of those cases that did not respond to conservative treatment. The causes of treatment failure in the remaining cases were a totally obstructed parotid duct and stenosis at the bifurcation site. CONCLUSION: Sialadenitis is the most common complication after RI therapy. Sialadenitis was successfully managed by conservative treatment in most cases, and interventional sialoendoscopy is an alternative method of treatment in selected cases such as in partial ductal stenosis.

Ethnicity, health and medical care: towards a critical realist analysis of general practice in the Korean community in Sydney.

This paper investigates the use and provision of biomedicine among Korean-Australian men on the basis of interview data from all of the eight Korean-speaking doctors practising in the Korean community in Sydney in 1995. From the viewpoint of these general practitioners, an analysis is made of the processes Korean men go through in adjusting to a new country, being involved in constant hard manual work and long working hours, and explores how they make use of all available resources to stay healthy. The Korean men have fully utilized the 'freely' available medical services under government-subsidized Medicare, bearing in mind that health is a capacity to work under the current environment, although illegal migrants restrained themselves from using it until they obtained legal status. Korean-speaking medical practitioners have been able to provide their fellow Koreans with 'culturally appropriate' health care, with the key factor being the absence of a language barrier. The level of patient satisfaction is high, possibly due to the excellent understanding the doctors have of the social aspects of illnesses, although the doctors do not go beyond curative medicine in their practice. However, the increasing number of Korean-speaking doctors in the small Korean community means that there is competition for patients. Consequently, the medical care is highly entrepreneurial. Referral by Korean doctors to practitioners of Korean herbal medicine is also a notable feature of the health care sector of the Korean community, especially as this offers Korean patients 'satisfactory' health relief for problems that are not easily relieved by doctors in the biomedical system.

Regulation of phospholipid synthesis in the yeast cki1Delta eki1Delta mutant defective in the Kennedy pathway. The Cho1-encoded phosphatidylserine synthase is regulated by mRNA stability.

In the yeast Saccharomyces cerevisiae, the most abundant phospholipid phosphatidylcholine is synthesized by the complementary CDP-diacylglycerol and Kennedy pathways. Using a cki1Delta eki1Delta mutant defective in choline kinase and ethanolamine kinase, we examined the consequences of a block in the Kennedy pathway on the regulation of phosphatidylcholine synthesis by the CDP-diacylglycerol pathway. The cki1Delta eki1Delta mutant exhibited increases in the synthesis of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine via the CDP-diacylglycerol pathway. The increase in phospholipid synthesis correlated with increased activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. However, other enzyme activities, including phosphatidylinositol synthase and phosphatidate phosphatase, were not affected in the cki1Delta eki1Delta mutant. For phosphatidylserine synthase, the enzyme catalyzing the committed step in the pathway, activity was regulated by increases in the levels of mRNA and protein. Decay analysis of CHO1 mRNA indicated that a dramatic increase in transcript stability was a major component responsible for the elevated level of phosphatidylserine synthase. These results revealed a novel mechanism that controls phospholipid synthesis in yeast.

Diacylglycerol pyrophosphate phosphatase in Saccharomyces cerevisiae.

Diacylglycerol pyrophosphate (DGPP) phosphatase in the yeast Saccharomyces cerevisiae is a Mg(2+)-independent and N-ethylmaleimide-insensitive 34-kDa vacuolar membrane-associated enzyme. It catalyzes the dephosphorylation of DGPP to form phosphatidate (PA) and then removes the phosphate from PA to form diacylglycerol (DAG). The enzyme is a member of the lipid phosphate phosphatase superfamily that contains a three-domain lipid phosphatase motif required for catalytic activity. Expression of the DPP1 gene, which encodes DGPP phosphatase, is induced by zinc depletion, by inositol supplementation, and when cells enter the stationary phase. Induction by zinc depletion is mediated by the transcription factor Zap1p, which binds to a zinc-responsive element in the DPP1 promoter. Repression of DPP1 expression is mediated by the transcription factor Gis1p, which binds to three post-diauxic shift elements in the promoter. Regulation of DPP1 correlates with the expression of DGPP phosphatase activity and the cellular levels of DGPP and PA.