Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).     

Novel and orally bioavailable inducible nitric oxide synthase inhibitors: synthesis and evaluation of optically active 4,5-dialkyl-2-iminoselenazolidine derivatives

We have previously reported that (4R,5R)-5-ethyl-2-imino-4-methylthiazolidine (3) strongly inhibits inducible nitric oxide synthase (iNOS). In a successive search for strong and selective iNOS inhibitors, we, herein, describe the synthesis of the selenium analogue of 3 (4: ES-2133) and its related optically active compounds and examine their in vitro and in vivo inhibitory activity against iNOS. In addition, an alternative synthetic method to the selected compound 4 and its pharmacokinetic profile is also reported.     

Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys⁷³ and, to the lesser extent, the active site Cys³². More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys⁷³, whereas Cys³² exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids and has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, post-ischemic reoxygenation injury, and aging (1). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injury and especially in the hepatic damage induced by several toxic substances. Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them is a variety of different aldehydes (2). The primary products of lipid peroxidation, lipid hydroperoxides, can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals giving rise to the formation of short chain, unesterified aldehydes, or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including protein, leading to disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by α,β-unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-alkenals, and 4-oxo-2-alkenals (3, 4).4-Hydroxy-2-nonenal (HNE),² among the reactive aldehydes, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (2, 5). HNE exerts these effects because of its facile reactivity with biological materials, particularly the sulfhydryl groups of proteins. The reaction of HNE with sulfhydryl groups leads to the formation of thioether adducts that further undergo cyclization to form cyclic hemiacetals (2). Although HNE also forms Michael adducts with the imidazole moiety of histidine residues and the ϵ-amino group of lysine residues (5), the formation of thiol-derived Michael adducts, stabilized as the cyclic hemiacetal, is considered to constitute the main reactivity of HNE, because of the nucleophilic potential of the sulfhydryl group compared with those of the imidazole and amine groups. However, because of the lack of specific and reliable methods for the determination of HNE-cysteine adducts, no study has so far quantitatively demonstrated their formation in proteins.Because HNE generated in lipid peroxidation is a racemic mixture of 4R- and 4S-enantiomers (6), the HNE Michael adducts, possessing three chiral centers at C-2, C-4, and C-5 in the tetrahydrofuran moiety (Fig. 1A), are composed of at least eight isomers. In our previous study (7), we characterized the configurational isomers of an HNE-histidine adduct by NMR spectroscopy and by molecular orbital calculations, and we found that the configuration of the tetrahydrofuran ring could affect the electron delocalization features, which contribute to the stability of the adduct. Moreover, we raised monoclonal antibodies against (R)-HNE- and (S)-HNE-histidine adducts and observed differential cellular distributions of these adducts in vivo. Balogh et al. (8) recently characterized the stereochemical configurations of the HNE-glutathione adduct by NMR experiments in combination with simulated annealing structure determinations. Despite these studies, however, the stereoselectivity of the HNE Michael addition adducts generated in proteins remains to be fully explored. In this study, to gain further structural insight into sulfhydryl modification by the lipid peroxidation product, we characterized the stereochemical configuration of the HNE-N-acetylcysteine adducts by NMR spectroscopy. In addition, we adapted a pyridylamination-based method for fluorescent labeling of the HNE-cysteine adducts, using 2-aminopyridine (2-AP) and sodium cyanoborohydride (NaCNBH₃), and successfully analyzed the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Furthermore, using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin (Trx).Open in a separate windowFIGURE 1.Reaction of cysteine residue with HNE. A, formation of the HNE-cysteine Michael adduct, possessing three chiral centers (asterisks). B, reaction of N-acetylcysteine with enantioisomeric HNE. The reactions were performed as described under “Experimental Procedures.” AU, absorbance units.     

3alpha/beta,20beta-hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH

Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.     

Conversion of a Chaperonin GroEL-independent Protein into an Obligate Substrate

Chaperones assist protein folding by preventing unproductive protein aggregation in the cell. In Escherichia coli, chaperonin GroEL/GroES (GroE) is the only indispensable chaperone and is absolutely required for the de novo folding of at least ∼60 proteins. We previously found that several orthologs of the obligate GroE substrates in Ureaplasma urealyticum, which lacks the groE gene in the genome, are E. coli GroE-independent folders, despite their significant sequence identities. Here, we investigated the key features that define the GroE dependence. Chimera or random mutagenesis analyses revealed that independent multiple point mutations, and even single mutations, were sufficient to confer GroE dependence on the Ureaplasma MetK. Strikingly, the GroE dependence was well correlated with the propensity to form protein aggregates during folding. The results reveal the delicate balance between GroE dependence and independence. The function of GroE to buffering the aggregation-prone mutations plays a role in maintaining higher genetic diversity of proteins.     

Prosaposin Overexpression following Kainic Acid-Induced Neurotoxicity

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.     

Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions

Analysis of bisulfite sequencing data usually requires two tasks: to call methylated cytosines (mCs) in a sample, and to detect differentially methylated regions (DMRs) between paired samples. Although numerous tools have been proposed for mC calling, methods for DMR detection have been largely limited. Here, we present Bisulfighter, a new software package for detecting mCs and DMRs from bisulfite sequencing data. Bisulfighter combines the LAST alignment tool for mC calling, and a novel framework for DMR detection based on hidden Markov models (HMMs). Unlike previous attempts that depend on empirical parameters, Bisulfighter can use the expectation-maximization algorithm for HMMs to adjust parameters for each data set. We conduct extensive experiments in which accuracy of mC calling and DMR detection is evaluated on simulated data with various mC contexts, read qualities, sequencing depths and DMR lengths, as well as on real data from a wide range of biological processes. We demonstrate that Bisulfighter consistently achieves better accuracy than other published tools, providing greater sensitivity for mCs with fewer false positives, more precise estimates of mC levels, more exact locations of DMRs and better agreement of DMRs with gene expression and DNase I hypersensitivity. The source code is available at http://epigenome.cbrc.jp/bisulfighter.     

Adipose-derived stem cells: Implications in tissue regeneration

Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differentiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs damaged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.