Atypical Biosynthetic Properties of a Δ12/ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Δ¹²-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C₁₈ substrates with ν+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (ν). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30°C versus 15°C). Thus, while maintaining the hallmark activity of the fatty acyl Δ¹²-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.Desaturases, the enzymes responsible for unsaturated fatty acid biosynthesis, are found throughout the eukaryotic taxa. Critical cellular processes dependent on the modification of acyl lipids by desaturases include the regulation of membrane structure and fluidity, proper function of ion channels and other membrane proteins, and the biosynthesis of signaling molecules, such as jasmonic acid and arachidonic acid-derived second messengers (53, 71). Polyunsaturated fatty acids (PUFAs) with double bonds at carbon-12, such as linoleic acid (18:2Δ9c,12c), are not synthesized by animals, who therefore depend upon the activities of the stepwise action of the Δ⁹- and Δ¹²-desaturases from plants and lower eukaryotes to generate these essential lipids.Supplementation of our diet with PUFAs derived from transgenic organisms has been targeted in recent years. Expression of fungal (37) and plant (56) desaturase genes in mammalian cells has been explored as a means to enhance the nutritional quality of meat products. Oleate and PUFA desaturases and elongases are gene targets sought after for transgenic production of the C₂₀ and C₂₂ polyunsaturated food supplements docosahexenoic and eicosapentenoic acids in alga, plants, and yeast (35, 51). The practical success of lipid metabolic engineering studies is dependent upon the expression of enzymes with high chemo- and regioselectivity within the transgenic organism, coupled with the manipulation of lipid biochemical flux to result in high, economically viable levels of unsaturated storage oil accumulation.Two evolutionarily distinct desaturase types exist: the soluble plastidal and the membrane-bound endoplasmic reticulum (ER)-localized enzymes, both of which use NAD(P)H and O₂ to sequentially abstract two hydrogens from vicinal sp³-hybridized carbons leading to a cis-alkene (15, 61). Current models for all fatty acyl desaturases postulate the activation of molecular oxygen at a nonheme diferrous active site that culminates with two C-H bond scissions and the formation of water (20, 29, 39, 46). In the case of the microsomal desaturases, conserved iron ligands appear to be located in three distinctive histidine box motifs (61). Microsomal Δ¹²-desaturases (FAD2s) are best known from plants, where they exhibit substantial (60 to 90%) sequence identity and have focused regioselectivity yet have evolved into the diverged desaturases that catalyze distinct oxidative processes, resulting in natural products with hydroxy, conjugated polyalkenyl, epoxy, and acetylenic functionalities (65). Studies of the FAD2 superfamily have been propelled by commercial interest in the modification of standard oilseed crops with “diverged” fad2 genes that show atypical regio- and/or chemoselectivity (32, 38, 50). Despite the sheer number of plant Δ¹²-desaturases, the refractory nature of these membrane-bound enzymes to purification has left structure/function relationships ill defined (33). Consequently, only the rough classification of the FAD2 enzymes into distinct functional or evolutionary classes has occurred, largely using genetic and in vivo functional characterization (12, 38, 42).While the Δ¹²- or FAD2 desaturases, which form a 12,13-double bond, are best known from plants, fungal fad2 homologs have been found in zygomycetes and ascomycetes (8, 52, 59). As suggested by molecular clock data, Basidiomycota and Ascomycotina diverged approximately 550 million years ago (3), indicating that metabolic basidiomycete genes may differ significantly from those of other fungal subtypes. With their high linoleic acid content, typically 60 to 80% of the lipid in basidiomycete fruiting bodies (63), and their ability to grow under varied temperature regimes, macrofungi provide an untapped genetic resource for desaturases that may be well suited for biotechnological applications. Indeed, two homobasidiomycete Δ¹²-desaturases have been recently reported (57, 77).In fungi, variations in membrane lipid composition caused by temperature cycling may be integral to the morphological changes of fruit body formation (58). Linoleate-derived hydroxy fatty acids and lactones have been shown to provide molecular signals, called Psi factors, involved in ascomycete sporulation (8, 9). Disruption of the oleoyl-phosphatidycholine desaturase odeA in Aspergillus parasiticus results in diminished growth; delayed germination has been proposed as a countermeasure for controlling this aflatoxigenic species (74). Additionally, volatile organic species emitted by fungi (e.g., (−)-1-octen-3-ol and 10-oxodecanoic acid) play a role in the palatability of mushrooms and may also mediate sporulation and the transition from vegetative to reproductive tissues (10). Separately, targeting Δ¹²-desaturases, which have no known homologs in humans, in pathogenic basidiomycetes has real potential as selective fungicidal targets. Cryptococcus neoformans infections in AIDS and immunosuppressed patients are frequently observed in the clinic; consequently, developing antimicrobial agents targeting C. neoformans will markedly improve the health of these patients (55).Phanerochaete chrysosporium is a widely distributed wood decay homobasidiomycete that has become a model system for studying lignocellulose degradation (41). It harbors an array of peroxidases and degrading lignocellulose as well as aromatic pollutants (14, 26). A role for linoleate (18:2), which may be supplied from endogenous wood lipids or through fungal Δ¹²-desaturation, in the mediation of lignin degradation has been suggested whereby diffusible lipid-derived peroxyl or alkoxy radicals aid in the initial decay of sound wood, particularly in white-rot fungi lacking lignin peroxidase (36, 73). The production of free 18:2 during early colonization of wood meal, followed by extracellular lipid peroxidation and in vitro degradation of nonphenolic lignin, has been shown for the white-rot fungus Ceriporiopsis subvermispora (16).As part of our program to elucidate the biosynthetic networks leading to highly unsaturated natural products in basidiomycetes (e.g., the polyacetylenes) (45), we carried out the cloning and sequence analysis of the gene encoding the sole Δ¹²-desaturase from P. chrysosporium. In this paper, we demonstrate its function through heterologous expression in Saccharomyces cerevisiae and show that this enzyme has features distinct from other fungal and plant FAD2 desaturases, which should facilitate future isolation and structure-function analysis of diverged macrofungal desaturases.     

Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro

Tyramine derivatives 3–27 were synthesized by using conventional and environmental friendly ultrasonic techniques. These derivatives were then evaluated for the first time for their α-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intestinal acetone powder) inhibitory activity by using in vitro mechanism-based biochemical assays. Compounds 7, 14, 20, 21 and 26 were found to be more active (IC₅₀?=?49.7?±?0.4, 318.8?±?3.7, 23.5?±?0.9, 302.0?±?7.3 and 230.7?±?4.0?μM, respectively) than the standard drug, acarbose (IC₅₀?=?840.0?±?1.73?μM (observed) and 780?±?0.028?μM (reported)) against α-glucosidase obtained from Saccharomyces cerevisiae. Kinetic studies were carried out on the most active members of the series in order to determine their mode of inhibition and dissociation constants. Compounds 7, 20 and 26 were found to be the competitive inhibitors of α-glucosidase. These compounds were also screened for their protein antiglycation, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Only compounds 20, 22 and 27 showed weak antiglycation activity with IC₅₀ values 505.27?±?5.95, 581.87?±?5.50 and 440.58?±?2.74?μM, respectively. All the compounds were found to be inactive against DDP-IV enzyme. Inhibition of α-glucosidase, DPP-IV enzymes and glycation of proteins are valid targets for the discovery of antidiabetic drugs. Cytotoxicity of compounds 3–27 was also evaluated by using mouse fibroblast 3T3 cell lines. All the compounds were found to be noncytotoxic. The current study describes the synthesis α-glucosidase inhibitory activity of derivatives, based on a natural product tyramine template. The compounds reported here may serve as the starting point for the design and development of novel α-glucosidase inhibitors as antidiabetic agents.     

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (< 90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced > 80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (< 90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

The circulatory small non-coding RNA landscape in community-acquired pneumonia on intensive care unit admission

Community-acquired pneumonia (CAP) is a major cause of sepsis. Despite several clinical trials targeting components of the inflammatory response, no specific treatment other than antimicrobial therapy has been approved. This argued for a deeper understanding of sepsis immunopathology, in particular factors that can modulate the host response. Small non-coding RNA, for example, micro (mi)RNA, have been established as important modifiers of cellular phenotypes. Notably, miRNAs are not exclusive to the intracellular milieu but have also been detected extracellular in the circulation with functional consequences. Here, we sought to determine shifts in circulatory small RNA levels of critically ill patients with CAP-associated sepsis and to determine the influence of clinical severity and causal pathogens on small RNA levels. Blood plasma was collected from 13 critically ill patients with sepsis caused by CAP on intensive care unit admission and from 5 non-infectious control participants. Plasma small RNA-sequencing identified significantly altered levels of primarily mature miRNAs in CAP relative to controls. Pathways analysis of high or low abundance miRNA identified various over-represented cellular biological pathways. Analysis of small RNA levels against common clinical severity and inflammatory parameters indices showed direct and indirect correlations. Additionally, variance of plasma small RNA levels in CAP patients may be explained, at least in part, by differences in causal pathogens. Small nuclear RNA levels were specifically altered in CAP due to Influenza infection in contrast to Streptococcus pneumoniae infection. Pathway analysis of plasma miRNA signatures unique to Influenza or Streptococcus pneumoniae infections showed enrichment for specific proteoglycan, cell cycle, and immunometabolic pathways.     

Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.     

Ultrastructural Analysis of ICP34.5? Herpes Simplex Virus 1 Replication in Mouse Brain Cells In Vivo

Replication-competent forms of herpes simplex virus 1 (HSV-1) defective in the viral neurovirulence factor infected cell protein 34.5 (ICP34.5) are under investigation for use in the therapeutic treatment of cancer. In mouse models, intratumoral injection of ICP34.5-defective oncolytic HSVs (oHSVs) has resulted in the infection and lysis of tumor cells, an associated decrease in tumor size, and increased survival times. The ability of these oHSVs to infect and lyse cells is frequently characterized as exclusive to or selective for tumor cells. However, the extent to which ICP34.5-deficient HSV-1 replicates in and may be neurotoxic to normal brain cell types in vivo is poorly understood. Here we report that HSV-1 defective in ICP34.5 expression is capable of establishing a productive infection in at least one normal mouse brain cell type. We show that γ34.5 deletion viruses replicate productively in and induce cellular damage in infected ependymal cells. Further evaluation of the effects of oHSVs on normal brain cells in animal models is needed to enhance our understanding of the risks associated with the use of current and future oHSVs in the brains of clinical trial subjects and to provide information that can be used to create improved oHSVs for future use.Several types of replication-competent neuroattenuated herpes simplex viruses (HSVs) are currently being evaluated in clinical cancer trials for safety and therapeutic activity (32), as well as for vaccine development (20). A critical safety concern associated with the clinical use of these oncolytic HSVs (oHSVs) is their ability to enter, replicate in, and spread to a wide range of cell types in different regions of the nervous system. One potential complication resulting from invasion of the central nervous system by HSV is herpes simplex encephalitis (HSE), an infection that causes lifelong neurological damage or death. A limited number of genes have been demonstrated to contribute to the virus''s ability to trigger HSE. The viral gene γ34.5 encodes the neurovirulence protein infected cell protein 34.5 (ICP34.5) (29). Viruses lacking the γ34.5 gene (e.g., R3616 and 1716) were found to be 5 logs less neurovirulent than wild-type strains of HSV-1 (4, 19, 36), as quantified by the intracranial LD₅₀, i.e., the lethal dose in 50% of mice inoculated intracerebroventricularly with the virus. The basis for this neuroattenuation was initially reported to be the inability of the γ34.5 deletion viruses to infect or replicate in brain cells (4). Subsequent immunohistochemical studies on infected brain tissue of intracerebroventricularly inoculated mice suggested that γ34.5 deletion viruses retained the ability to infect a wide range of brain cell types and to replicate in and, by day 7, destroy ependymal cells (ECs) (16, 21).To create a more neuroattenuated and thus safer virus, the virus G207 was constructed from the γ34.5 deletion virus R3616 by insertional mutagenesis of the U_L39 gene (25). The U_L39 gene encodes the large subunit of the viral ribonucleotide reductase (vRR) (29). Cellular ribonucleotide reductase is a DNA synthetic enzyme which is of low abundance in quiescent cells but is critical for the synthesis of DNA precursors and is thus abundant in mitotically active cells such as cancer cells. Based on the phenotype of viruses mutated in the vRR alone (13), this double-deletion virus lacking both ICP34.5 and vRR expression is predicted to restrict viral replication to cancer cells expressing cellular RR at levels sufficient to support viral replication (25). In preclinical studies with mice, inoculation with G207 via the intracerebroventricular route failed to destroy the EC layer at 5 days postinoculation (34). These studies supported the concept that a double-deletion virus may be safer in clinical trials than a virus lacking only ICP34.5 expression.To test the hypothesis that productive replication of γ34.5 deletion viruses is restricted to cancer cells, we developed sensitive methods to examine the ability of γ34.5 deletion viruses, with either intact or mutated vRR, to replicate productively in vivo and to complete the multistep process of virion assembly and egress.Common to most models of HSV virion assembly and egress is the observation that capsid proteins translated in the cytoplasm are imported to the nucleus, where a capsid shell is assembled and viral DNA is subsequently packaged. Capsids containing viral DNA are distinguished by an electron-dense (dark) center, whereas capsids lacking viral DNA contain a core protein visible by electron microscopy (EM) often as an inner concentric circle. In subsequent steps, DNA-filled capsids acquire an envelope by budding through the inner nuclear membrane into the perinuclear space. Capsids observed between the inner and outer nuclear membranes have an envelope and tegument and resemble mature extracellular virions (10).Consensus is lacking on the specific sequence of subsequent stages of viral egress, and multiple pathways may exist (3, 18, 24, 30). In the subsequent step of the envelopment-deenvelopment-reenvelopment model (18, 30), enveloped capsids in the perinuclear space lose their envelope by fusion with the outer nuclear membrane as the capsids enter the cytoplasm. In this model, progeny viruses are thus present in the cytoplasm as naked capsids. Cytoplasmic naked capsids acquire their mature envelope as they bud into a cytoplasmic organelle (e.g., a Golgi body).According to an alternative model, enveloped capsids move within the perinuclear space into the endoplasmic reticulum (ER), which is continuous with the perinuclear space (33). From this space, enveloped capsids, individually or in groups, bud off within a vesicle membrane characteristic of the outer nuclear membrane/ER. Within these vesicles, enveloped virions are transported through the cytoplasm. In a final step common to both models, the cytoplasmic vesicle releases mature enveloped virions into the extracellular space by fusing with the cell membrane.ECs are an ideal cell type for these studies due to their distinct morphology and location (described below) and their reported function as neural stem cells (15). We reasoned that since mitotic activity is the reported basis for the productive replication and selectivity of γ34.5 deletion viruses in cancer cells (9, 34), and ECs may be mitotically active, if any normal brain cell type were to support productive replication of γ34.5 deletion viruses, ECs would be the most likely candidate.ECs line the cerebral ventricles, acting as a semipermeable barrier between the brain parenchyma and the cerebrospinal fluid (CSF) in the ventricles (7, 12). Their location thus makes them easily exposed to the virus via intraventricular injections. Their location, combined with their morphologically distinct cuboid shape with kinocilia and microvilli that protrude into the CSF, allows them to be easily excised and recognized under both light microscopy and EM.Here we report the results of a side-by-side comparative study evaluating whether a double-deletion virus similar to G207 and a virus lacking only ICP34.5 expression differ from each other and from a wild-type virus in the ability to infect and replicate productively in ECs of the mouse brain in vivo. The results of these studies are consistent with results of other studies in that they demonstrate that viruses similar to those used in clinical trials (e.g., G207, HSV1716) have a greatly attenuated ability to replicate compared to that of a wild-type virus. However, our data also show very clearly that γ34.5 deletion viruses do replicate productively in infected mouse brain ECs in vivo. These studies suggest that (i) ECs can serve as an exquisitely sensitive model for future evaluations of the ability of oHSVs to replicate productively in normal mouse brain cells and (ii) the potential exists for double-deletion oHSVs to damage normal brain cells. Thus, further comparative studies are warranted to determine whether this risk is sufficiently high to restrict the administration of ICP34.5 deletion viruses in or near the cerebral ventricles in clinical studies.     

A Novel MMP-2 Inhibitor 3-azidowithaferin A (3-azidoWA) Abrogates Cancer Cell Invasion and Angiogenesis by Modulating Extracellular Par-4

BackgroundWithaferin A, which is a naturally derived steroidal lactone, has been found to prevent angiogenesis and metastasis in diverse tumor models. It has also been recognized by different groups for prominent anti-carcinogenic roles. However, in spite of these studies on withanolides, their detailed anti-metastatic mechanism of action remained unknown. The current study has poised to address the machinery involved in invasion regulation by stable derivative of Withaferin A, 3-azido Withaferin A (3-azidoWA) in human cervical HeLa and prostate PC-3 cells.Conclusion/SignificanceFor this report, we found that 3-azidoWA suppressed motility and invasion of HeLa and PC-3 cells in MMP-2 dependent manner. Our in vitro result strongly suggests that sub-toxic doses of 3-azidoWA enhanced the secretion of extracellular Par-4 that abolished secretory MMP-2 expression and activity. Depletion of secretory Par-4 restored MMP-2 expression and invasion capability of HeLa and PC-3 cells. Further, our findings implied that 3-azidoWA attenuated internal phospho-ERK and phospho-Akt expression in a dose dependent manner might play a key role in inhibition of mouse angiogenesis by 3-azidoWA.     

Knowledge,Attitudes and Practices of Contraception among Afghan Refugee Women in Pakistan: A Cross-Sectional Study

Background

During the 1980s, approximately three million people migrated from Afghanistan to Pakistan and sought refuge in several cities including the city of Karachi. After the initial settlement of the refugees, the international organizations transitioned the health care of these refugees to the two local non-profit service agencies in Karachi. One of these agencies subsidized health care to the refugees under their care and the other agency encouraged the refugees under their care to utilize governmental and non-governmental private health resources at the disposal of general public. Our objective was to measure the effect of health subsidy on the uptake of contraception among Afghan refugee women and compare them to the group of Afghan women without such a subsidy.

Methodology/Principal Findings

A randomly selected group of 650 married Afghan women-325 women in each group-participated in a detailed survey regarding the knowledge, attitude and practices of family planning and contraceptive use. 90 percent of the women in the health subsidy group had had heard of family planning, compared to the 45 percent in the non-subsidized group. The use of contraceptives was greater than two-fold in the former versus the latter. Results of logistic regression analysis revealed that the refugee women who had had access to subsidized healthcare were significantly more likely to use the contraceptive methods with advancing age as compared to the women in the non-health subsidy group. The difference remained significant after adjusting for other variables.

Conclusions/Significance

Refugee women who are provided subsidized healthcare are more inclined to use contraceptives. It is therefore important that Afghan refugee women living elsewhere in Pakistan be provided healthcare subsidy, whereby their reproductive health indicators could improve with reduced fertility. We strongly encourage facilities introducing such subsidies to refugees in resource poor settings to assess the impact through similar inquiry.