Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Differential loss of enzyme activity by vitC and iron containing proteins

Our earlier studies showed that rabbit muscle phosphoglucomutase was irreversibly inactivated by exposure to a mixture of vitamin C, FeCl3 and O2. The enzyme lost about 70% of its phosphate (V.V. Desphande and J.G. Joshi, J. Biol. Chem. 260, 754-764, 1985). The present report shows that several other iron proteins can substitute for FeCl3 to a varying degree. The rate of inactivation by FeCl3 greater than ferritin greater than hemoglobin = hemerythrin greater than transferrin = ferridoxin = vitamin C. These iron compounds also produced dephosphoenzyme but did not dephosphorylate ATP, ADP, AMP or phospholipids.     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Production of L-Phenylacetyl Carbinol by Immobilized Cells of Saccharomyces Cerevisiae

Conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC) was achieved with immobilized, growing cells of Saccharomyces cerevisiae in different reactors. Product formation increased (31%) with the subsequent initial reuses of the entrapped cells. Biomass production and PAC formation depleted (40 and 57%, respectively) after 4-5 continuous growth and biotransformation cycles. With the regeneration of the biocatalysts, catalytic activity of the cells was resumed. The highest yields were in a stirred tank reactor (29 g PAC) from 77 g benzeldehyde with 14 repeated uses of entrapped cells.     

Involvement of reactive oxygen species in the oxidation of tyrosine and dopa to melanin and in skin tanning

The role of reactive oxygen (1O2 and O2-.) in skin photosensitization and tanning reaction has been examined. Riboflavin (RF), hematoporphyrin (HP), 3-carbethoxypsoralen (3-CP), and 8-methoxypsoralen (8-MOP), upon photoexcitation under aerobic conditions, produced singlet O2 (1O2). RF, 3-CP, and 8-MOP also produced superoxide anion (O2-.). Reactive O2 produced by photosensitized RF, 3-CP, and 8-MOP was found to oxidize tyrosine and dopa to dopachrome and subsequently their conversion to melanin. HP did not oxidize tyrosine to dopachrome, and 3-CP and RF revealed substantial oxidation of tyrosine. Dopa was oxidized to dopachrome and subsequently to melanin by all photosensitizers tested at a variable rate as follows: RF greater than 3-CP greater than HPD greater than 8-MOP. UVA alone and to a lesser extent UVB also produced 1O2 which induced the oxidation of tyrosine and dopa to dopachrome and subsequently to melanin. The production of dopachrome was higher with dopa compared to tyrosine under all irradiation conditions. These observations appear to have relevance to the O2-requiring immediate tanning reaction of the skin stimulated by solar radiation and in the induction of skin photosensitization.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin–eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion’s trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco’s modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients’ needs.     

Genome sequencing of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> (NRCC1), a novel isolate from dromedary camel (<Emphasis Type="Italic">Camelus dromedarius</Emphasis>) rumen fluid

The lactic acid bacterium Pediococcus acidilactici has recently been reported to help in treating constipation, diarrhea, relieving stress, and enhancing growth rate and immune response in humans, birds, fishes, and small animals. In the present study, we sequenced and analyzed the whole genome of P. acidilactici NRCC1, a novel isolate from rumen fluid of dromedary camel (Camelus dromedarius). The genome of P. acidilactici NRCC1 was assembled into 60 contigs, comprising 1,785,679 bp and 42.5% GC content. The 1705 CDS were predicted and annotated using the RAST server. The genome encodes numerous enzymes for utilization of different carbohydrates. It also harbors genes for antibiotic biosynthesis and many others which might confer probiotic properties. The comparative genome analysis with P. acidilactici DSM 20284 revealed some unique features in P. acidilactici NRCC1. Thus, the genome sequencing of P. acidilactici NRCC1 has opened up new horizons for further research in animal probiotics and feed supplements.