Extracts from Argentinian native plants reverse fluconazole resistance in Candida species by inhibiting the efflux transporters Mdr1 and Cdr1.

BACKGROUND: The development of multidrug resistance (MDR) associated with the overexpression of the efflux transporters Mdr1 and Cdr1 in Candida species impedes antifungal therapies. The urgent need for novel agents able to inhibit the function of both pumps, led us to evaluate this property in 137 extracts obtained from Argentinian plants. METHODS: The ability of the extracts to reverse efflux pump-mediated MDR was determined with an agar chemosensitization assay using fluconazole (FCZ) resistant Mdr1- and Cdr1-overexpressing clinical isolates of Candida albicans and Candida glabrata as well as Saccharomyces cerevisiae strains selectively expressing Mdr1 (AD/CaMDR1) or Cdr1 (AD/CaCDR1). The resistance-reversing activity of the most potent extracts was further confirmed using a Nile Red accumulation assay. RESULTS: Fifteen plant extracts overcame the FCZ resistance of Candida albicans 1114, which overexpresses CaMdr1 and CaCdr1, and AD/CaMDR1, with those from Acalypha communis and Solanum atriplicifolium being the most effective showing 4- to 16-fold reversal of resistance at concentrations ≥ 25 µg/mL. Both extracts, and to a lesser extent that from Pterocaulon alopecuroides, also restored FCZ sensitivity in CgCdr1-overexpressing C. glabrata 109 and in AD/CaCDR1 with fold reversal values ranging from 4 to 32 and therefore demonstrating a dual effect against Mdr1 and Cdr1. Both, A. communis and S. atriplicifolium extracts at concentrations ≥ 12.5 and ≥ 25 µg/mL, respectively, increased the intracellular Nile Red accumulation in all yeast strains overexpressing efflux pumps. CONCLUSIONS: The non-toxic and highly active extracts from A. communis and S. atripicifolium, provide promising sources of compounds for potentiating the antifungal effect of FCZ by blocking the efflux function of Mdr1 and Cdr1 transporters.

Complex patterns of circulating fatty acid levels in gestational diabetes mellitus subclasses across pregnancy.

BACKGROUND & AIMS: To investigate the relationship between maternal serum fatty acid levels and gestational diabetes mellitus (GDM) subtypes across pregnancy. METHODS: A total of 680 singleton mothers enrolled in the Complex Lipids in Mothers and Babies (CLIMB) study in Chongqing, China were included. Clinical information and serum samples were collected at gestational weeks (GWs) 11-14, 22-28, and 32-34. 75 g Oral Glucose Tolerance Test (OGTT) was conducted at GW 24-28 and GDM subtypes divided into three groups using International Association of Diabetes and Pregnancy Study Group (IADPSG) guidelines criteria: elevated fasting plasma glucose (FPG group; n = 59); 1-h and/or 2-h post-load glucose (1h/2h-PG group; n = 94); combined group (FPG&1h/2h-PG group; n = 42). Non-GDM pregnancies were included (n = 485) as controls. Twenty fatty acids were quantified in serum using gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: Overall, most serum fatty acid concentrations increased rapidly from the first to second trimester, followed by a plateauing or reduction in the third trimester (p < 0.001). In cross sectional analysis, fatty acid concentrations were significantly higher in the FPG group at GW 11-14 and decreased in the 1h/2h-PG group at GW 32-34, relative to controls. Moreover, higher α-linolenic acid (ALA; the second tertile: adjusted odds ratio [aOR] = 2.53, 95% CI: 1.17 to 5.47; the third tertile: aOR = 2.60, 95% CI: 1.20 to 5.65) and docosahexaenoic acid (DHA; the second tertile: aOR = 2.34, 95% CI: 1.10 to 4.97; the third tertile: aOR = 2.16, 95% CI: 1.00 to 4.63) were significantly associated with a higher risk of GDM in women with elevated fasting plasma glucose at GW 11-14 (first tertile as reference). CONCLUSIONS: Our findings highlight the importance of considering GDM subtypes for the individualised management of GDM in pregnancy. ALA and DHA in early pregnancy are associated with a higher risk of FPG-GDM subtype. This has widespread implications when recommending n-3 PUFAs supplementation for women with GDM.

Response of human periodontal ligament cells to baicalin.

BACKGROUND: Periodontitis is the most common cause of tooth loss in adults. Periodontal ligament cell (PLC)-based therapy is considered one of the most promising methods in periodontal tissue regeneration. The traditional Chinese medicine baicalin has been shown to possess antimicrobial and anti-inflammatory activities and enhance cell proliferation and alkaline phosphatase activity. The aim of this study is to investigate the response of human PLCs (HPLCs) to baicalin. METHODS: The effect of baicalin on cultured HPLC proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of baicalin on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), core binding factor α1 (Cbfα1), and osteocalcin (OC) was determined by quantitative real-time polymerase chain reaction and immunodetection. RESULTS: Baicalin at a concentration of 0.01 µg/mL promoted HPLC proliferation, upregulated OPG messenger RNA (mRNA) and protein expression, and downregulated RANKL mRNA and protein expression. In addition to reducing the RANKL/OPG expression ratio significantly, it also increased Cbfα1 and OC mRNA and protein expression. CONCLUSION: Baicalin showed multifaceted regulation of genes with important roles in tissue growth and differentiation, and thus it has the potential to be a promising candidate for HPLC-based periodontal regeneration therapy.

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.

Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.

Antifungal saponins from Paris polyphylla Smith.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

Amino acid residues affecting drug pump function in Candida albicans--C. albicans drug pump function.

Membrane-located drug transporters are important components in the multidrug resistance of microbial cells and human tissues. In fungi, clinically important resistance to antifungal drugs most often results from the over-expression of efflux pump proteins in the plasma membrane of the resistant cell. This review describes studies of the ATP binding cassette (ABC) family of membrane efflux pumps in the opportunistic human pathogen Candida albicans and, in particular, examines how changes in the polypeptide sequence can affect pump function. The identification of amino acid residues affecting pump function can provide new insights into efflux pump mechanisms and the relationship between structure and function. Such information will be important for the design of pump inhibitors which could supplement existing antifungal drugs.