The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4 degrees C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5-10 microM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.

Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.

Activation of cGMP-dependent protein kinase (PKG) has antitumor effects in colon cancer cells but the mechanisms are not fully understood. The present study has examined the regulation of β-catenin/TCF signaling, since this pathway has been highlighted as central to the antitumor effects of PKG. We show that PKG activation in SW620 cells results in reduced β-catenin expression and a dramatic inhibition of TCF-dependent transcription. PKG did not affect protein stability, nor did it increase phosphorylation of the amino-terminal Ser33/37/Thr41 residues that are known to target β-catenin for degradation. However, we found that PKG potently inhibited transcription from a luciferase reporter driven by the human CTTNNB1 promoter, and this corresponded to reduced β-catenin mRNA levels. While PKG was able to inhibit transcription from both the CTNNB1 and TCF reporters, the effect on protein levels was less consistent. Ectopic PKG had a marginal effect on β-catenin protein levels in SW480 and HCT116 but was able to inhibit TCF-reporter activity by over 80%. Investigation of alternative mechanisms revealed that cJun N-terminal kinase (JNK) activation was required for the PKG-dependent regulation of TCF activity. PKG activation caused β-catenin to bind to FOXO4 in colon cancer cells, and this required JNK. Activation of PKG was also found to increase the nuclear content of FOXO4 and increase the expression of the FOXO target genes MnSOD and catalase. FOXO4 activation was required for the inhibition of TCF activity since FOXO4-specific siRNA completely blocked the inhibitory effect of PKG. These data illustrate a dual inhibitory effect of PKG on TCF activity in colon cancer cells that involves reduced expression of β-catenin at the transcriptional level, and also β-catenin sequestration by FOXO4 activation.

Analysis of knockout animals indicates that 30 ,5 0 cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2 þ / þ and Prkg2 À / À mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2 þ / þ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2 þ / þ but not Prkg2 À / À mice. Treatment of Prkg2 À / À mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.

In recent years, several antitumor signaling pathways mediated by the cGMP-dependent protein kinases have been identified in colon cancer cells. This review aims to present the mounting evidence in favor of cGMP/protein kinase G (PKG) signaling as a therapeutic strategy in colon cancer. The homeostatic and tumor suppressive effects of cGMP in the intestine are uncontested, but the signaling details are not understood. PKG is the central cGMP effector, and can block proliferation and tumor angiogenesis by inhibiting β-catenin/TCF and SOX9 signaling. Therapeutic activation of cGMP/PKG offers a promising avenue for the prevention and treatment of colon cancer, but additional preclinical studies are needed to fully understand the potential of this system.

Microbial production of desired compounds provides an efficient framework for the development of renewable energy resources. To be competitive to traditional chemistry, one requirement is to utilize the full capacity of the microorganism to produce target compounds with high yields and turnover rates. We use integrated computational methods to generate and quantify the performance of novel biosynthetic routes that contain highly optimized catalysts. Engineering a novel reaction pathway entails addressing feasibility on multiple levels, which involves handling the complexity of large‐scale biochemical networks while respecting the critical chemical phenomena at the atomistic scale. To pursue this multi‐layer challenge, our strategy merges knowledge‐based metabolic engineering methods with computational chemistry methods. By bridging multiple disciplines, we provide an integral computational framework that could accelerate the discovery and implementation of novel biosynthetic production routes. Using this approach, we have identified and optimized a novel biosynthetic route for the production of 3HP from pyruvate. Biotechnol. Bioeng. 2012; 109:572–582. © 2011 Wiley Periodicals, Inc.

Endocannabinoids play essential roles in synaptic plasticity; thus, their dysfunction often causes impairments in memory or cognition. However, it is not well understood whether deficits in the endocannabinoid system account for the cognitive symptoms of schizophrenia. Here, we show that endocannabinoid-mediated synaptic regulation is impaired by the prolonged elevation of neuregulin-1, the abnormality of which is a hallmark in many patients with schizophrenia. When rat hippocampal slices were chronically treated with neuregulin-1, the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids, was enhanced due to the increased expression of its degradative enzyme, monoacylglycerol lipase. As a result, the time course of depolarization-induced 2-AG signaling was shortened, and the magnitude of 2-AG-dependent long-term depression of inhibitory synapses was reduced. Our study reveals that an alteration in the signaling of 2-AG contributes to hippocampal synaptic dysfunction in a hyper-neuregulin-1 condition and thus provides novel insights into potential schizophrenic therapeutics that target the endocannabinoid system.

Abstract. This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (~53% vs ~68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (~47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (~20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (~15%) than did the non-delipated oocytes, whether centrifuged or not (~4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, ~39% were activated and male pronucleus formation was observed in ~40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.