Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Addressing PXR liabilities of phthalazine-based hedgehog/smoothened antagonists using novel pyridopyridazines

Pyridopyridazine antagonists of the hedgehog signaling pathway are described. Designed to optimize our previously described phthalazine smoothened antagonists, a representative compound eliminates a PXR liability while retaining potency and in vitro metabolic stability. Moreover, the compound has improved efficacy in a hedgehog/smoothened signaling mouse pharmacodynamic model.     

Synthesis and structure–activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel,potent PPARα selective activators- PPARα and PPARγ selectivity modulation

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARα/γ dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARα versus PPARγ. Potent, highly selective PPARα activators 2a and 2l, as well as PPARα activators with significant PPARγ activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.     

Telocytes accompanying cardiomyocyte in primary culture: two- and three-dimensional culture environment

Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.     

Engineered heart tissue graft derived from somatic cell nuclear transferred embryonic stem cells improve myocardial performance in infarcted rat heart

The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT‐ES]) cells to treat diseases. Nevertheless, it is controversial as NT‐ES cells may pose risks in their therapeutic application. EHT from NT‐ES cell‐derived cardiomyocytes was generated through a series of improved techniques in a self‐made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2–4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT‐ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT‐ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Development of a Common Oligonucleotide Reference Standard for Microarray Data Normalization and Comparison across Different Microbial Communities

High-density functional gene arrays have become a powerful tool for environmental microbial detection and characterization. However, microarray data normalization and comparison for this type of microarray remain a challenge in environmental microbiology studies because some commonly used normalization methods (e.g., genomic DNA) for the study of pure cultures are not applicable. In this study, we developed a common oligonucleotide reference standard (CORS) method to address this problem. A unique 50-mer reference oligonucleotide probe was selected to co-spot with gene probes for each array feature. The complementary sequence was synthesized and labeled for use as the reference target, which was then spiked and cohybridized with each sample. The signal intensity of this reference target was used for microarray data normalization and comparison. The optimal amount or concentration were determined to be ca. 0.5 to 2.5% of a gene probe for the reference probe and ca. 0.25 to 1.25 fmol/μl for the reference target based on our evaluation with a pilot array. The CORS method was then compared to dye swap and genomic DNA normalization methods using the Desulfovibrio vulgaris whole-genome microarray, and significant linear correlations were observed. This method was then applied to a functional gene array to analyze soil microbial communities, and the results demonstrated that the variation of signal intensities among replicates based on the CORS method was significantly lower than the total intensity normalization method. The developed CORS provides a useful approach for microarray data normalization and comparison for studies of complex microbial communities.Microarray-based technology has become a robust genomic tool to detect, track, and profile hundreds to thousands of different microbial populations simultaneously in complex environments such as soils and sediments. For example, GeoChip, a comprehensive functional gene array, has been developed for investigating biogeochemical, ecological, and environmental processes (12, 18, 23, 27, 29, 32). Although a massive amount of microarray data can be generated rapidly, one of the bottlenecks in using microarrays for environmental microbial community studies is the lack of an appropriate standard for data comparison and normalization (6). Currently, it is difficult to compare microarray data across different sites, experiments, laboratories, and/or time periods (10). This limits the power of the technology to address ecological and environmental questions.In pure culture-based functional genomics studies, genomic DNAs (gDNAs) have been used as a common reference for hybridizations in which the same amount of gDNAs are used to cohybridize with each target cDNA sample and then to normalize different target cDNAs based on the gDNA standard (4, 5, 8, 9, 19, 21, 23). Several normalization methods such as scale normalization, quantile normalization, and Lowess normalization have been used for gene expression studies (2). Using the gDNA standard method can minimize or eliminate differences in target cDNA quantity, spot morphology, uneven hybridization, labeling, and sequence-specific hybridization behaviors (5), and this allows the comparison of microarray data across different sites, laboratories, experiments, and/or times. The main rationale for gDNA as a common reference is that it provides complete coverage for all genes represented on the array because the DNA composition from a particular organism should be identical across different treatment samples even though RNA expression is different (8). However, this approach is not applicable to microbial community studies because not all communities have identical DNA compositions. Pooling of equal amounts of gDNA or RNA from every target sample to make a common sample could be used as an alternative reference for cohybridization (1, 22). However, the disadvantage of the sample pooling approach is that samples do not provide large amounts of DNA or RNA in a reliable and reproducible way. For example, groundwater samples usually have a very low biomass and thus would not provide enough DNA for pooling. In addition, the sample pool itself is uncharacterized, and gene abundance may be diluted out so that insufficient DNA is present to result in a positive signal some array features, especially for those genes in low abundance. Moreover, a new sample pool would be required for every new experiment, making comparison across experiments difficult. Thus, other approaches need to be developed for microbial community studies.Dudley et al. (7) used a 25-mer oligonucleotide that matched a small portion of the parental EST clone vector contained in every PCR product printed on the array for normalization of pure culture RNA expression. Although the oligonucleotide generated a stable hybridization signal on every array feature, this method requires a universal sequence tag as a “capture” sequence, limiting its general use in microbial community studies. Thus, in the present study, we developed a common oligonucleotide reference standard (CORS) approach by co-spotting a common oligonucleotide with each array feature to improve the accuracy and comparability of microarray data for microbial community studies. This method was evaluated by using a pilot array, a whole-genome array, and a functional gene array, and all results demonstrate that the developed CORS is a reliable and reproducible method for microarray data normalization and comparison for microbial community studies.     

Effects of two species of inorganic arsenic on the nutrient physiology of rice seedlings

This paper reports on a hydroponics experiment that was conducted to investigate the effect of inorganic arsenics on the seedlings of the rice cultivar Shanyou63. The seedlings were subjected to two treatments, i.e., As(III) and As(V). The results showed that the morphological traits of the seedlings were significantly altered after the arsenic treatments. Analysis of nitrogen, phosphorus, potassium, and arsenic contents of the roots and leaves of the seedlings indicated that the absorption of phosphorus and potassium was mainly affected by As(III), while that of nitrogen was mainly affected by As(V). The expression of 12 genes involved in the absorption and utilization of nitrogen, phosphorus, and potassium were all observed to be down-regulated after the arsenic treatments. As(V) significantly affected the absorption and utilization of nitrogen, while As(III) significantly affected those of phosphorus and potassium. The result obtained by real-time FQ-PCR regarding the difference in the gene expressions agreed with that of our hydroponics experiment.     

Development and characterization of microsatellite loci in Taihangia rupestris (Rosaceae), a rare cliff herb

? Premise of the study: Microsatellite primers were developed for the rare Taihangia rupestris (Rosaceae) to evaluate genetic diversity, population genetic structure, mating system, and demographic events of this species. ? Methods and Results: Ten primer sets were developed using an enriched genomic library and were successfully amplified in T. rupestris var. ciliata and T. rupestris var. rupestris. The number of alleles per locus ranged from 2 to 21; the observed and expected heterozygosities ranged from 0.300 to 0.950 and from 0.328 to 0.956, respectively, in the two varieties. ? Conclusions: The markers described here will be useful for studies of genetic variation, genetic structure, and mating systems of T. rupestris, which are important for the future conservation of this rare species.