The Antifungal Agent Itraconazole Induces the Accumulation of High Mannose Glycoproteins in Macrophages

Bacterial lipopolysaccharide (LPS) is a key mediator in the development of Gram-negative septic shock, which is a major health problem. The effect of LPS on myeloid cells is mediated by a multicomplex receptor system in which CD14, a glycosylphosphatidylinositol-anchored glycoprotein, and Toll-like receptor 4 are the major players. We have found that incubation of macrophages with itraconazole (ICZ), an azole antifungal commonly used in humans, altered both the expression and glycosylation of CD14. This glycoprotein, which is endo H-resistant in untreated cells, becomes endo H-sensitive following ICZ treatment. The effect of ICZ on glycan processing was observed in all newly synthesized glycoproteins as indicated by incorporation of [2-³H]mannose. In addition, cells treated with ICZ increased surface concanavalin A (ConA) binding, corroborating an increase in high mannose surface glycoproteins. Although the glycosylation pattern of CD14 was altered, this glycoprotein was delivered to the cell surface or was secreted. Moreover, it appeared functional as demonstrated by the release of LPS-induced tumor necrosis factor-α under conditions specific for a CD14-mediated activation process. The effect of ICZ on glycosylation was not dependent on inhibition of the cholesterol biosynthetic pathway and was specific for this drug because other azole antifungals, such as ketoconazole and econazole, did not alter glycan processing. These results suggest a possible secondary effect of ICZ that impacts the processing of glyconjugates and may alter cellular function and homeostasis.Bacterial lipopolysaccharide (LPS),² or endotoxin, is a component of the outer membrane of Gram-negative bacteria and induces a robust inflammatory response. LPS is a trigger for Gram-negative sepsis, which is a major heath problem in the United States (1, 2). LPS is a pathogen-associated molecular pattern and is recognized by specific surface receptors on myeloid cells. The major LPS binding site on macrophages is CD14, which is a glycosylphosphatidylinositol-anchored, leucine-rich repeat glycoprotein (3). The recognition of LPS by CD14 requires that the bacterial product associates first with LPS-binding protein. Then, the complex of CD14 and LPS interacts with Toll-like receptor 4 and MD-2 to initiate the release of cytokines and other effector molecules (4). The importance of CD14 in LPS signaling is illustrated by the fact that it enhances the recognition of LPS up to 1000-fold (5). Moreover, CD14-deficient mice are LPS insensitive and resistant to septic shock (6). In addition to the glycosylphosphatidylinositol-anchored membrane CD14 (mCD14), a soluble variant (sCD14) of this glycoprotein is found in the extracellular space and circulation.We have previously found that the expression of mCD14 in macrophages (Mϕs) is enhanced by lovastatin, which is a statin widely used for the treatment of hypercholesteremia. This elevation in CD14 expression was not correlated with a decrease in cellular cholesterol levels, but, rather, it was partially dependent on the depletion of geranylgeranylpyrophosphate (7), which is a non-sterol isoprenoid product of the cholesterol biosynthetic pathway. These observations illustrate that the statin-mediated inhibition of cholesterol biosynthesis has several additional side effects. This pathway is responsible for the synthesis of several important cellular products, such as the isoprenoid intermediates, which are involved in the post-translational modification of proteins. Consequently, the effect of statins on cellular physiology needs to be confirmed by using additional inhibitors for other steps in the cholesterol biosynthetic pathway. In an effort to understand if endogenously synthesized cholesterol affects CD14 expression, we used itraconazole (ICZ), which inhibits the cytochrome P450 (CYP450) enzyme 14α-demethylase, a key step in the conversion of lanosterol to cholesterol. This step in the cholesterol pathway is downstream of the isoprenoid intermediates, avoiding the inhibitory effects on isoprenoid moieties. We found that treatment of Mϕs with ICZ altered the processing of glycoproteins from high mannose to complex structures. Thus, cells treated with ICZ displayed an enrichment of high mannose glycoproteins on the surface. Moreover, this effect of ICZ was independent of the cholesterol pathway and unique to this drug. This dramatic alteration in glycosylation may have a tremendous impact on cellular function and may be pertinent to patients taking ICZ.     

Polyploidy creates higher diversity among Cynodon accessions as assessed by molecular markers

Developing a better understanding of associations among ploidy level, geographic distribution, and genetic diversity of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to: (1) determine ploidy analysis of Cynodon accessions collected from Turkey, (2) investigate associations between ploidy level and diversity, (3) determine whether geographic and ploidy distribution are related to nuclear genome variation, and (4) correlate among four nuclear molecular marker systems for Cynodon accessions’ genetic analyses. One hundred and eighty-two Cynodon accessions collected in Turkey from an area south of the Taurus Mountains along the Mediterranean cost and ten known genotypes were genotyped using sequence related amplified polymorphism (SRAP), peroxidase gene polymorphism (POGP), inter-simple sequence repeat (ISSR), and random amplified polymorphic DNA (RAPD). The diploids, triploids, tetraploids, pentaploids, and hexaploids revealed by flow cytometry had a linear present band frequency of 0.36, 0.47, 0.49, 0.52, and 0.54, respectively. Regression analysis explained that quadratic relationship between ploidy level and band frequency was the most explanatory (r = 0.62, P < 0.001). The AMOVA results indicated that 91 and 94% of the total variation resided within ploidy level and provinces, respectively. The UPGMA analysis suggested that commercial bermudagrass cultivars only one-third of the available genetic variation. SRAP, POGP, ISSR, and RAPD markers differed in detecting relationships among the bermudagrass genotypes and rare alleles, suggesting more efficiency of combinatory analysis of molecular marker systems. Elucidating Cynodon accessions’ genetic structure can aid to enhance breeding programs and broaden genetic base of commercial cultivars. O. Gulsen and S. Sever-Mutlu contributed equally to this work.     

Green light effects on biological systems: a new biophysical phenomenon

This paper reports a new phenomenon connected with the influence of green light (GL) on biological systems. Our experiments have revealed an antioxidant effect of GL on cells subjected to lethal doses of UV at the cellular level and a protective effect of GL on DNA denatured by UV, coupled with a structural modification of DNA macromolecules under GL irradiation, at the molecular level. Mouse melanocyte cultures are subjected to UV irradiations with L₅₀ fluxes of 16.0 J m^− 2 s^− 1. GL is obtained from a strontium aluminate pigment, which emits GL under UV activation. Cells grown in GL, prior to UV irradiation, present a clear surprising protective effect with surviving values close to the controls. A GL antioxidant effect is suggested to be mediated through GL influence on cellular water cluster dynamics. To test this hypothesis, reactive oxygen species (ROS) are determined in cell cultures. The results revealed a decrease of cellular ROS generation in the UV-irradiated samples protected by a previous 24 h of GL irradiation. At the DNA level, the same type of GL protection against UV damage is recorded by gel electrophoresis and by UV spectroscopy of the irradiated DNA molecules. Two physical methods, impedance spectroscopy and chronoamperometry, have revealed at the level of GL-irradiated DNA molecules spectral modifications that correlate with the UV spectroscopy results. The interaction between the chargeless photons and the field of water molecules from the cellular compartments is discussed in relation with the new field of macroscopic quantum coherence phenomena.     

Ethnomedical Syndromes and Treatment-Seeking Behavior among Mayan Refugees in Chiapas,Mexico

This survey investigated the prevalence of ethnomedical syndromes and examined treatments and treatment-seeking in Mayan Guatemalans living in United Nations High Commissioner for Refugee (UNHCR) camps in Chiapas, Mexico. Methods included a rapid ethnographic assessment to refine survey methods and inform the cross-sectional survey, which also examined mental health outcomes; 183 households were approached for interview, representing an estimated 1,546 residents in five refugee camps and 93% of all households. One adult per household (N = 170) was interviewed regarding his or her health; an additional 9 adults in three surveyed households participated and were included in this analysis; of the 179 participants, 95 primary child-care providers also answered a children’s health questionnaire for their children. Results indicated that ethnomedical syndromes were common in this sample, with 59% of adults and 48.4% of children having experienced susto (fright condition) and 34.1% of adults reporting ataques de nervios (nervous attacks); both conditions were significantly associated with symptoms consistent with posttraumatic stress disorder, anxiety and depression and are mental health conditions recognized by the American Psychiatric Association. Combining healthcare provider and indigenous treatments such as physician prescribed medication (65%), medicinal plants (65.7%), and limpias (spiritual cleansings) (40.6%) was reported. Most participants (86%) sought routine medical treatment from UNHCR trained health promoters in their camp. Assessing ethnomedical health is important for informing mental health programs among this population.     

The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction

Arginine-specific ADP-ribosyltransferase (Art) catalyzes the mono-ADP-ribosylation, in which it transfers a single ADP-ribose moiety of NAD to the arginine residue(s) of target proteins, and may regulate the function of the proteins or peptides in cellular processes. In vertebrates, Art family is consisted of seven members (Arts1-7), and these Arts are distributed among various tissues except B lymphocytes. Previously, we described molecular cloning, characterization and distribution of glycosylphosphatidylinositol (GPI)-anchored Arts, Art7.1 and Art7.2 (formerly, we referred as cgArt1 and cgArt2, respectively) in chicken tissues (Terashima et al (2005) Biochem J 389:853-861). Here, we demonstrate for the first time that Art7.1 was predominantly expressed on the surface of B cells from the bursa of Fabricius as a GPI-anchored form, as well as on T cells from the thymocytes. Furthermore, we show that the expression of Art7.1 molecules on B cells could modulate the B cell receptor (BCR) signalling and direct the B cell fate to maturation. Thus, our present observation sheds light on the Art molecule expressed on B cells and its possible functional role in BCR signalling.     

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.     

Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.