Controlling selectivity and enhancing yield of flavonoid glycosides in recombinant yeast.

Flavonoid glycosides are known for their medicinal properties and potential use as natural sweeteners. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase from Dianthus caryophyllus was used as a whole-cell biocatalyst. The yeast system's performance was characterized using the flavanone naringenin as a model substrate for the production of naringenin glycosides. It was found that final naringenin glycoside yields increased in a dose-dependent manner with increasing initial naringenin substrate concentrations. However, naringenin concentrations >0.5 mM did not give further enhancements in glycoside yield. In addition, a method for controlling overall selectivity was discovered where the glucose content in the culture medium could be altered to control the selectivity, making either naringenin-7-O-glucoside (N7O) or naringenin-4'-O-glucoside (N4O) the major products. The highest yields achieved were 87 mg/L of N7O and 82 mg/L of N4O using 40MSGI and 2xMSGI media, respectively. The effects of two intermediates involved in UDP-glucose biosynthesis, uridine 5'-monophosphate (UMP) and orotic acid, on glycoside yields were also determined. Addition of UMP to the culture medium significantly decreased glycoside yield. In contrast, addition of orotic acid to the culture medium significantly enhanced the glycoside yield and shifted the selectivity toward N7O. The highest naringenin glycoside yield achieved using 10 mM orotic acid in the 40MSGI media was 155 mg/L, a 71% conversion of substrate to product.

MMP-28 as a regulator of myelination.

BACKGROUND: Matrix metalloproteinase-28 (MMP-28) is a poorly understood member of the matrix metalloproteinase family. Metalloproteinases are important mediators in the development of the nervous system and can contribute to the maturation of the neural micro-environment. RESULTS: MMP-28 added to myelinating rat dorsal root ganglion (DRG) co-cultures reduces myelination and two antibodies targeted to MMP-28 (pAb180 and pAb183) are capable of binding MMP-28 and inhibiting its activity in a dose-dependent manner. Addition of 30 nM pAb180 or pAb183 to rat DRG cultures resulted in the 2.6 and 4.8 fold enhancement of myelination respectively while addition of MMP-28 to DRG co-cultures resulted in enhanced MAPK, ErbB2 and ErbB3 phosphorylation. MMP-28 protein expression was increased within demyelinated lesions of mouse experimental autoimmune encephalitis (EAE) and human multiple sclerosis lesions compared to surrounding normal tissue. CONCLUSION: MMP-28 is upregulated in conditions of demyelination in vivo, induces signaling in vitro consistent with myelination inhibition and, neutralization of MMP-28 activity can enhance myelination in vitro. These results suggest inhibition of MMP-28 may be beneficial under conditions of dysmyelination.

Enhanced cell cycle progression and down regulation of p21(Cip1/Waf1) by PRL tyrosine phosphatases.

Human PRL-1, PRL-2, and PRL-3 tyrosine phosphatases induce the malignant transformation of epithelial cells. We tested the hypothesis that the oncogenic effects of PRL occur by increasing cellular proliferation. Cells stably transfected with PRL-1 or PRL-2 exhibited 2.7-3.3-fold increases over control cells in the rate of DNA synthesis and the proportion of cells in S-phase, and they progressed more rapidly from G1 into S. In addition, cells overexpressing either PRL-1 or PRL-2 exhibited enhanced cyclin-dependent kinase 2 (CDK2) activity and significantly lower p21(Cip1/Waf1) protein levels, and PRL-1 overexpressing cells had higher cyclin A protein levels than control cells. We conclude that PRL phosphatases increase cell proliferation by stimulating progression from G1 into S phase, and this process may be dependent on the down regulation of the cyclin dependent kinase inhibitor p21(Cip1/Waf1).

Effects of the isoprenoids perillyl alcohol and farnesol on apoptosis biomarkers in pancreatic cancer chemoprevention.

Perillyl alcohol, farnesol and geraniol have chemotherapeutic activity toward pancreatic and other cancers. Perillyl alcohol induces apoptosis and increases expression of the proapoptotic protein Bak in cultured pancreatic tumor cells. We tested the hypothesis that farnesol and geraniol would have similar effects. After 48 hours of treatment with farnesol geraniol or perillyl alcohol, human BxPC3 pancreatic cancer cells exhibited a 3 to 10-fold increase in apoptosis and higher Bak expression than the controls. We then tested the hypotheses that perillyl alcohol and farnesol would have chemopreventive activity toward pancreatic cancer and would increase Bak expression and apoptosis in vivo. Hamster pancreatic cancer was initiated at time 0 with N-nitrosobis(2-oxopropyl)amine. Animals were fed control, 2% (w/w) perillyl alcohol, or 1% (w/w) farnesol diets from weeks 5-42. Pancreatic carcinoma incidence was decreased by perillyl alcohol and farnesol. Hyperplastic pancreatic ductal neoplasms from perillyl alcohol and farnesol-treated animals had higher Bak protein expression (p < 0.05), and somewhat higher apoptotic rates, diminished expression of the antiapoptotic protein BCL-XL, and lower rates of DNA synthesis than the controls.

Tissue-specific alterations of PRL-1 and PRL-2 expression in cancer.

The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of these molecules in human neoplasms is not well understood. To increase understanding of the role PRL-1 and PRL-2 play in the neoplastic process, in situ hybridization was used to examine PRL-1 and PRL-2 mRNA expression in 285 normal, benign, and malignant human tissues of diverse origin. Immunohistochemical analysis was performed on a subset of these. PRL-1 and PRL-2 mRNA expression was also assessed in a small set of samples from a variety of diseases other than cancer. Where possible, associations with clinicopathological characteristics were evaluated. Alterations in PRL-1 or -2 expression were a frequent event, but the nature of those alterations was highly tumor type specific. PRL-1 was significantly overexpressed in 100% of hepatocellular and gastric carcinomas, but significantly under-expressed in 100% of ovarian, 80% of breast, and 75% of lung tumors. PRL-2 expression was significantly increased in 100% of hepatocellular carcinomas, yet significantly downregulated in 54% of kidney carcinomas. PRL-1 expression was correlated to patient gender in the bladder and to patient age in the brain and skeletal muscle. PRL-1 expression was also associated with tumor grade in the prostate, ovary, and uterus. These results suggest a pleiotropic role for PRL-1 and PRL-2 in the neoplastic process. These molecules may associate with tumor progression and serve as clinical markers of tumor aggressiveness in some tissues, but be involved in inhibition of tumor formation or growth in others.

Proteomic analysis of demyelinated and remyelinating brain tissue following dietary cuprizone administration.

Cuprizone intoxication is a commonly used model of demyelination that allows the temporal separation of demyelination and remyelination. The underlying biochemical alterations leading to demyelination, using this model, remain unclear and may be multifold. Analysis of proteomic changes within the brains of cuprizone-exposed animals may help elucidate key cellular processes. In the current study, we report the results of the liquid chromatography tandem mass spectrometry analysis of total protein from the brain hemispheres of control and toxin-exposed mice at 6 weeks of exposure and after 3 and 6 weeks of recovery to identify protein changes during the remyelination phase. We found that at 6 weeks of cuprizone exposure, myelin proteins were reduced compared to controls and increased throughout the course of recovery, as expected. In contrast, other protein groups, such as proteins related to mitochondrial function, were increased at 6 weeks of treatment compared to untreated controls and returned toward control levels following withdrawal of toxin. These results suggest that a global proteomic analysis of the brain tissue of cuprizone-treated mice can identify changes related to the demyelination/remyelination process.

Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry.

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.

Cell cycle arrest by the isoprenoids perillyl alcohol, geraniol, and farnesol is mediated by p21(Cip1) and p27(Kip1) in human pancreatic adenocarcinoma cells.

Pancreatic cancer, the fourth leading cause of cancer-associated mortality in the United States, usually presents in an advanced stage and is generally refractory to chemotherapy. As such, there is a great need for novel therapies for this disease. The naturally derived isoprenoids perillyl alcohol, farnesol, and geraniol have chemotherapeutic potential in pancreatic and other tumor types. However, their mechanisms of action in these systems are not completely defined. In this study, we investigated isoprenoid effects on the cell cycle and observed a similar antiproliferative mechanism of action among the three compounds. First, when given in combination, the isoprenoids exhibited an additive antiproliferative effect against MIA PaCa-2 human pancreatic cancer cells. Furthermore, all three compounds induced a G(0)/G(1) cell cycle arrest that coincided with an increase in the expression of the cyclin kinase inhibitor proteins p21(Cip1) and p27(Kip1) and a reduction in cyclin A, cyclin B1, and cyclin-dependent kinase (Cdk) 2 protein levels. Immunoprecipitation studies demonstrated increased association of both p21(Cip1) and p27(Kip1) with Cdk2 as well as diminished Cdk2 kinase activity after isoprenoid exposure, indicating a cell cycle-inhibitory role for p21(Cip1) and p27(Kip1) in pancreatic adenocarcinoma cells. When siRNA was used to inhibit expression of p21(Cip1) and p27(Kip1) proteins in MIA PaCa-2 cells, conditional resistance to all three isoprenoid compounds was evident. Given similar findings in this cell line and in BxPC-3 human pancreatic adenocarcinoma cells, we conclude that the chemotherapeutic isoprenoid compounds perillyl alcohol, farnesol, and geraniol invoke a p21(Cip1)- and p27(Kip1)-dependent antiproliferative mechanism in human pancreatic adenocarcinoma cells.

Neural MMP-28 expression precedes myelination during development and peripheral nerve repair.

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment.

RECQL4-deficient cells are hypersensitive to oxidative stress/damage: Insights for osteosarcoma prevalence and heterogeneity in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a heterogeneous disease, associated with increased prevalence of osteosarcoma in very young patients with a mutated RECQL4 gene. In this study, we tested the ability of RECQL4 deficient fibroblasts, derived from a RTS patient to recover from hydrogen peroxide (H(2)O(2))-induced oxidative stress/damage. Immunoperoxidase staining for 8-oxo-deoxyguanosine (8-oxo-dG) formation in RTS and normal human fibroblasts were compared to assess DNA damage. We determined DNA synthesis, cell growth, cell cycle distribution, and viability in RTS and normal human fibroblasts before and after H(2)O(2) treatment. H(2)O(2) induces 8-oxo-dG formation in both RTS and normal fibroblasts. In normal human fibroblasts, RECQL4 was predominantly localized to cytoplasm; nuclear translocation and foci formation occurred in response to oxidant stimulation. After recovery from oxidant exposure, viable RTS fibroblasts showed irreversible growth arrest compared to normal fibroblasts. DNA synthesis decreased significantly in treated RTS cells, with concomitant reduction of cells in the S-phase. These results suggest that enhanced oxidant sensitivity in RECQL4 deficient fibroblasts derived from RTS patients could be attributed to abnormal DNA metabolism and proliferation failure. The ramifications of these findings on osteosarcoma prevalence and heterogeneity in RTS are discussed.