Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus

Objective: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic Psammomys obesus using novel electron microscopy technologies. Background: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle. Methods: Assessment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy X-ray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemius muscle. Results: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls. Conclusions: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.     

Rapid Methods for High-Throughput Detection of Sulfoxides

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.The growing demand for green catalytic processes has increased the utilization of enzymes as industrial biocatalysts for the synthesis of fine chemicals (6, 19, 20). As a consequence, there is a continuous search for novel or improved biocatalysts. In order to find an appropriate candidate for a process, various sources of enzymes must be screened for activity (23). Therefore, a sensitive, reproducible, accurate, and simple high-throughput screening method is a key prerequisite for the development of biocatalytic processes on an industrial scale (32, 39).Screening systems are divided into three different classes. The first class contains assays applicable to testing growing or resting microbial colonies for enzymatic activity directly on agar plates (23), for example, detection of epoxide hydrolase activity on butane oxide by use of safranin O. Oxidation of the 1,2-diol product by Escherichia coli modified the membrane potential and led to accumulation of the red dye in the colonies producing active enzyme (34). In another study, the spontaneous oxidation of substituted catechols to brown-red quinones was used to screen random libraries of whole cells expressing toluene monooxygenases (TMOs) for regioselective oxidation of substituted phenols (12, 30). The positive clones produced a red halo around the cells. These assays are high-throughput, simple procedures but often require a tailored substrate with a chromophore, such as bromonaphthol or azo-dye (23).The second class includes chromogenic and fluorogenic assays applicable in microtiter plates or microarray formats (23). Microtiter plates in 96- or 384-well format are particularly well suited for spectroscopic reading using either UV-visible or fluorescence plate readers. This class may be subdivided into the following four groups: (i) enzyme-coupled assays, such as the determination of dehydrogenase activity through formation of NADH from NAD and an absorbance change at a wavelength of 340 nm; (ii) assays using chromogenic and fluorogenic substrates, such as various synthetically labeled substrates that are commercially available for the determination of hydrolytic activity produced by lipases, phosphatases, glycosidases, amidases, etc.; (iii) assays using chromogenic and fluorogenic sensors, such as widely used pH indicators (16), that may be applied in any reaction that includes a change in pH; and (iv) microarray assays using a solid support, enabling screening of thousands of samples. The high-throughput potential of these methods was demonstrated by profiling of 40 different esterases and lipases across 35 different fluorogenic ester substrates, using only 50 μl of each enzyme solution and a submilligram quantity of each substrate for over 7,000 tests (2).The third class of enzymatic assays rely on product detection by instruments and include gas chromatography (GC), high-pressure liquid chromatography (HPLC), mass spectrometry, nuclear magnetic resonance (NMR) spectrometry, and infrared radiation assays that have been adapted for high throughput (22, 23, 33). Such assays require expensive and sophisticated equipment, but they allow working directly with the substrate of interest and are rapidly adapted once the instrument is available (23).Various chemical substances can be synthesized by bacteria and fungi, among which are the chiral sulfoxides (5, 10, 11, 24, 36). As natural products, chiral sulfoxides possess a wide range of biological activities, from flavor and aroma precursor activities to antimicrobial properties. In addition, they are efficient auxiliaries that lead to essential asymmetric transformations (3, 11). Furthermore, one of the most significant applications of chiral sulfoxides is in the pharmaceutical industry (3). The world''s best-selling antiulcer drug, (S)-omeprazole, is a chiral sulfoxide (11, 14). Although there have been numerous reports on chemical and biological methods for synthesizing chiral sulfoxides, little information exists about rapid high-throughput assays for sulfoxide determination. In this study, four colorimetric or fluorometric procedures were evaluated and adapted for screening of whole-cell libraries containing variants of TMOs. Three of the four methods were exploited successfully to a high-throughput format using 96-well microtiter plates, whereas one method was not suitable due to low sensitivity. The method based on acid activation of omeprazole proved very efficient, but no positive variants were found, whereas the one based on selective inhibition of horse liver alcohol dehydrogenase (HLADH), originally reported by Sprout and Seto (28), was useful for detecting mutants with high activity and enantioselectivity in the oxidation of methyl p-tolyl sulfide.     

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development

1. Download : Download high-res image (125KB)
2. Download : Download full-size image

     

Physiological relevance of successive hydroxylations of toluene by toluene para-monooxygenase of Ralstonia pickettii PKO1

We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m-cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (V_max/K_m=0.046, 0.036, and 0.055 mL min^-1 mg^-1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta-cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.     

BAF-1 mobility is regulated by environmental stresses

Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.     

Sexual and asexual development: two distinct programs producing the same tunicate

1. Download : Download high-res image (216KB)
2. Download : Download full-size image

     

SARS-CoV-2 spike variants exhibit differential infectivity and neutralization resistance to convalescent or post-vaccination sera

1. Download : Download high-res image (186KB)
2. Download : Download full-size image