The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate Methanol Metabolism antagonistically in pichia pastoris

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5' CYCCNY 3' motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.

Improvement in Bone Properties by Using Risedronate Adsorbed Hydroxyapatite Novel Nanoparticle Based Formulation in a Rat Model of Osteoporosis

A superior drug formulation capable of achieving efficient osteogenesis is in imperative demand for the treatment of osteoporosis. In the present study we investigated the potential of using novel risedronate-hydroxyapatite (HA) nanoparticle based formulation in an animal model of established osteoporosis. Nanoparticles of HA loaded with risedronate (NHLR) of various sizes (80-130 nm) were generated for bone targeted drug delivery. Three months after ovariectomy, 36 ovariectomized (OVX) rats were divided into 6 equal groups and treated with various doses of NHLR (500,350 and 250 mu g/kg intravenous single dose) and sodium risedronate (500 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. One month after drug administration, the left tibia and femur were tested for bone mechanical properties and histology, respectively. In the right femur, bone density was measured by method based on Archimedes principle and bone porosity analyses were performed using X-ray imaging. NHLR (250 mu g/kg) showed a significant increase in bone density and reduced bone porosity when compared with OVX control. Moreover, NHLR (250 mu g/kg) significantly increased bone mechanical properties and bone quality when compared with OVX control. The results strongly suggest that the NHLR, which is a novel nanoparticle based formulation, has a therapeutic advantage over risedronate sodium monotherapy for the treatment of osteoporosis in a rat model of postmenopausal osteoporosis.

Cytochrome P450-mediated metabolism in brain: functional roles and their implications

Introduction: Cytochromes P450 (P450) and associated monooxygenases are a family of heme proteins involved in metabolism of endogenous compounds (arachidonic acid, eicosanoids and prostaglandins) as also xenobiotics including drugs and environmental chemicals. Liver is the major organ involved in P450-mediated metabolism and hepatic enzymes have been characterized. Extrahepatic organs, such as lung, kidney and brain have the capability for biotransformation through P450 enzymes. Brain, including human brain, expresses P450 enzymes that metabolize xenobiotics and endogenous compounds. Areas covered: An overview of P450-mediated metabolism in brain is presented focusing on distinct differences seen in expression of P450 enzymes, generation of unique P450 enzymes in brain through alternate splicing and their consequences in terms of metabolism of psychoactive drugs and inflammatory prompts, such as leukotrienes, thus modulating inflammatory response. Expert opinion: The brain possesses unique P450s that metabolize drugs and endogenous compounds through pathways that are markedly different from that seen in liver indicating that extrapolation directly from liver to brain is not appropriate. It is therefore necessary to characterize the unique brain P450s and their ability to metabolize xenobiotics and endogenous compounds to better understand the functions of this important class of enzymes in brain, especially human brain.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2 A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1 alpha, IL-1 beta, G-CSF, GM-CSF, MIP-1 alpha, IFN-gamma, IL-17, and TNF-alpha. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality (C) 2013 Elsevier Inc. All rights reserved.

Scaffolds for bone tissue engineering: role of surface patterning on osteoblast response

The fabrication of tissue engineering scaffolds necessitates amalgamation of a multitude of attributes including a desirable porosity to encourage vascular invasion, desired surface chemistry for controlled deposition of calcium phosphate-based mineral as well as ability to support attachment, proliferation, and differentiation of lineage specific progenitor cells. Scaffold fabrication often includes additional surface treatments to bring about desired changes in the surface chemistry. In this perspective, this review documents the important natural and synthetic scaffolds fabricated for bone tissue engineering applications in tandem with the surface treatment techniques to maneuver the biocompatibility of engineered scaffolds. This review begins with a discussion on the fundamental concepts related to biocompatibility as well as the characteristics of the biological micro-environment. The primary focus is to discuss the effects of surface micro/nano patterning on the modulation of bone cell response. Apart from reviewing a host of experimental studies reporting the functionality of osteoblast-like bone cells and stem cells on surface modified or textured bioceramic/biopolymer scaffolds, theoretical insights to predict cell behavior on a scaffold with different topographical features are also briefly analyzed.

Methionine down-regulates TLR4/MyD88/NF-kappa B signalling in osteoclast precursors to reduce bone loss during osteoporosis

Background and PurposeStudies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss. Experimental ApproachThe anti-resorptive effect of methionine, (250mgkg(-1) body wt administered in drinking water for 10 weeks), was evaluated in ovariectomized (OVX) rats by monitoring changes in bone turnover, formation of osteoclasts from blood-derived mononuclear cells and changes in the synthesis of pro-osteoclastogenic cytokines. Key resultsMethionine improved bone density and significantly decreased the degree of osteoclast development from blood mononuclear cells in OVX rats, as indicated by decreased production of osteoclast markers tartarate resistant acid phosphatase b (TRAP5b) and MIP-1. siRNA-mediated knockdown of myeloid differentiation primary response 88 MyD88], a signalling molecule in the toll-like receptor (TLR) signalling cascade, abolished the synthesis of both TRAP5b and MIP-1 in developing osteoclasts. Methionine supplementation disrupted osteoclast development by inhibiting TLR-4/MyD88/NF-B pathway. Conclusions and ImplicationsTLR-4/MyD88/NF-B signalling pathway is integral for osteoclast development and this is down-regulated in osteoporotic system on methionine treatment. Methionine treatment could be beneficial for the treatment of postmenopausal osteoporosis.