Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.     

Gallic acid induces apoptosis in 3T3-L1 pre-adipocytes via a Fas- and mitochondrial-mediated pathway

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound. Our previous studies have shown that some phenolic acids such as gallic acid inhibit cell growth and induce apoptosis in 3T3-L1 pre-adipocytes. However, the molecular mechanism of gallic acid in the induction of cell apoptosis is still unclear. In this study, we investigated the effect of gallic acid on the apoptotic pathway in 3T3-L1 pre-adipocytes. Western blot data revealed that gallic acid stimulated an increase in the protein expression of Fas, FasL, and p53. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was changed by gallic acid treatment. Gallic acid released mitochondrial cytochrome c into the cytosol and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase. Pretreatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented gallic acid from inhibiting cell viability in 3T3-L1 pre-adipocytes. The data also indicated that treatment with gallic acid inhibited histone deacetylase activity in 3T3-L1 pre-adipocytes. These results demonstrate that gallic acid induces apoptosis in 3T3-L1 pre-adipocytes through the Fas and mitochondrial pathway. The induction of cell apoptosis by gallic acid may prove to be a pivotal mechanism for decreased pre-adipocyte proliferation.     

Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes

Obesity has become a global epidemic in both developed and developing countries, and it is a significant risk factor for various diseases such as diabetes, cancer, heart disease, and hypertension. In the present study, the effect of naturally occurring antioxidants (flavonoids and phenolic acids) on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. The results showed that o-coumaric acid and rutin had the highest inhibition on intracellular triglyceride (61.3 and 83.0%, respectively) among 15 phenolic acids and 6 flavonoids tested. However, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 adipocytes was not influenced by those compounds. For glycerol-3-phosphate dehydrogenase (GPDH) activity, the data indicated that o-coumaric acid and rutin had the highest inhibition on GPDH activity (54.2 and 66.8%, respectively) among the compounds tested. o-Coumaric acid and rutin also inhibited the expression of PPARgamma, C/EBPalpha and leptin and then up-regulated expression of adiponectin at the protein level. Some naturally occurring antioxidants efficiently suppressed adipogenesis in 3T3-L1 adipocytes. These results suggest that o-coumaric acid and rutin targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome.     

Effect of prepubertal feeding regimen on reproductive development of gilts.

The effect of prepubertal feed level on growth and reproductive development of gilts was investigated. At 13 wk. of age, white crossbred gilts were penned individually and assigned to the following treatments: Ad lib, ad libitum intake from 13 to 25 wk. of age (n = 64); Control, ad libitum intake from 13 wk. of age until 100 kg BW and then 90% of ad libitum intake until 25 wk. of age (n = 65); and Restricted, 74% of ad libitum intake from 13 wk. to 25 wk. of age (n = 64). Feed was formulated to primarily restrict energy intake. The study was replicated in two seasons. At 25 wk. of age, gilts were moved to group pens, approximately 16 gilts/pen, allowed ad libitum access to feed, and estrus detection was initiated. Gilts were mated at first estrus and those recycling were remated. After mating, gilts were moved to gestation stalls and fed 1.5x maintenance. At 30 d of gestation, reproductive tracts were harvested, and numbers of corpora lutea (CL) and live embryos were recorded. From 13 to 25 wk. of age, feed consumption was 258 for Ad lib, 251 for Control, and 189 kg/gilt for Restricted, and, from 13 wk. of age until 30 d of gestation, total feed consumption was 367 for Ad lib, 356 for Control, and 299 kg/gilt for Restricted gilts. Age at puberty (196 d) and pregnancy (200 d) was not affected (P>.18) by treatment. However, the rate at which gilts attained puberty (e.g., percentage pubertal at 28 d) was greatest in Ad lib (75) and least in Control (61) gilts. Number of CL and live embryos at 30 d of gestation/gilt assigned to the study was unaffected (P>.21) by treatment. Quantity of feed consumed from 13 wk. of age to 30 d of gestation per live embryo in gilts assigned to the study was 40.0 for Ad lib, 39.8 for Control, and 30.6 kg/gilt for Restricted gilts. These results indicate that moderate feed restriction of gilts during prepubertal development may increase efficiency of swine production without negative impact on reproductive performance through 30 d of gestation.     

Genomic Relationships of Triticum sharonense with Other S-Genome Diploid Triticum Species

Triticum sharonense was hybridized with autotetra-ploid T. speltoides, T. longissimum and T. bicorne. Meiotic analysis of these hybrids showed that T. sharonense is almost equally related to both T. speltoidcs and T. longissimum, while it is comparatively distant from T. bicorne. Therefore, this study does not support treating T. sharonense as a subspeies or variety of T. lottgissimum.     

Sources of resistance to head scab in Triticum

Multi-floret and single-floret injection inoculation methods were used to test 1076 accessions of Triticum for resistance to initial infection and resistance to pathogen spread within spike tissue respectively. The data obtained between years or inoculation dates were comparatively similar and were little influenced by climatic factors, especially humid condition. All the tested materials uniformly exhibited susceptibility to initial infection, but there were great differences in resistance to spread among species or cultivars. The diploid and tetraploid wheats were severely susceptible. Only 30 genotypes of common wheat of T. aestivum concv. vulgare showed high resistance to spread. The highest frequency of high spread resistance existed in these landraces which were distributed in Zhejiang, Hubei, Hunan, Jiangsu provinces and Shanghai City, Guizhou, Sichuan and Yunnan provinces ranked second. The majority of wheat landraces from Hebei, Shanxi, Shaanxi Province and Beijing City showed susceptibility or high susceptibility. No resistance was found in Tibetian wheats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antioxidant activity of phenolic compounds isolated from Mesona procumbens Hemsl

The antioxidant activity of phenolic compounds isolated from Mesona procumbens Hemsl. (Hsian-tsao) was investigated. Hsian-tsao was extracted with various solvents, and the results showed that the fraction treated with acidic ethyl acetate (pH 2) possessed large amounts of phenolic compounds and a strong antioxidant activity on peroxidation of linoleic acid. The antioxidant activity (inhibition of peroxidation, IP%) of the acidic ethyl acetate of Hsian-tsao extract at 50 microg/mL (98.9%) was stronger than those of 50 microg/mL alpha-tocopherol (78%) and BHA at 10 microg/mL (90%). When fractionated with Amberlite XAD-7 gel chromatography, the acidic ethyl acetate fraction of Hsian-tsao extract was separated into four subfractions (A-D). Subfraction B, with high yield and strong antioxidant activity, was further isolated and purified and then identified as containing protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, and syringic acid by means of UV, EI-MS, and (1)H and (13)C NMR. The antioxidant capability of isolated compounds was also determined using the thiocyanate system and the erythrocyte ghost system. The results indicate that the phenolic acids could be important antioxidant components in Hsian-tsao, among which caffeic acid with the highest antioxidant activity and the greatest content is most important.     

Protective effect of Mesona procumbens against tert-butyl hydroperoxide-induced acute hepatic damage in rats

The protective effect of Hsian-tsao (Mesona procumbens Hemsl.) and its active compounds on liver damage was evaluated using the model of tert-butyl hydroperoxide (t-BHP)-induced acute hepatic damage in rats. Male Sprague-Dawley rats (200 +/- 10 g) were orally pretreated with a water extract of Hsian-tsao (WEHT) (0.1, 0.5, and 1.0 g/kg) or caffeic acid (0.1 g/kg of body weight) for 13 days before a single dose of t-BHP (0.2 mmol/kg, intraperitoneally) to each animal, and the rats were sacrificed 18 h later by decapitation; blood samples were collected for the assays of serum biochemical values. The livers were excised from the animals and assayed for oxidative injury, antioxidant enzyme, and pathological histology. The result showed that the oral pretreatment of WEHT (0.1, 0.5, and 1.0 g/kg) or caffeic acid (0.10 g/kg) before t-BHP (0.2 mmol/kg) treatment significantly lowered the serum levels of the hepatic enzyme markers (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase) and reduced oxidative stress of the liver by evaluation of malondialdehyde, glutathione, 8-hydroxy-2'-deoxyguanosine, glutathione peroxidase, and glutathione reductase. The histopathological evaluation of the rat livers showed that WEHT and caffeic acid reduced the incidence of liver lesions including cloudy swelling, pyknosis, and cytolysis induced by t-BHP in rats. On the basis of the results of this study, it can be speculated that M. procumbens protects liver against t-BHP-induced hepatic damage in rats.     

Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts

The objective of this study was to investigate the inhibitory effect of naturally occurring flavonoids on individual stage of protein glycation in vitro using the model systems of delta-Gluconolactone assay (early stage), BSA-methylglyoxal assay (middle stage), BSA-glucose assay, and G.K. peptide-ribose assay (last stage). In the early stage of protein glycation, luteolin, qucertin, and rutin exhibited significant inhibitory activity on HbA1C formation (p < 0.01), which were more effective than that of aminoguanidine (AG, 10 mM), a well-known inhibitor for advanced glycation endproducts (AGEs). For the middle stage, luteolin and rutin developed more significant inhibitory effect on methylglyoxal-medicated protein modification, and the IC50's were 66.1 and 71.8 microM, respectively. In the last stage of glycation, luteolin was found to be potent inhibitors of both the AGEs formation and the subsequent cross-linking of proteins. In addition, phenyl-tert-butyl-nitron served as a spin-trapping agent, and electron spin resonance (ESR) was used to explore the possible mechanism of the inhibitory effect of flavonoids on glycation. The results indicated that protein glycation was accompanied by oxidative reactions, as the ESR spectra showed a clear-cut radical signal. Statistical analysis showed that inhibitory capability of flavonoids against protein glycation was remarkably related to the scavenging free radicals derived from glycoxidation process (r = 0.79, p < 0.01). Consequently, the inhibitory mechanism of flavonoids against glycation was, at least partly, due to their antioxidant properties.     

Evidence that the reactivity of the martian soil is due to superoxide ions

The Viking Landers were unable to detect evidence of life on Mars but, instead, found a chemically reactive soil capable of decomposing organic molecules. This reactivity was attributed to the presence of one or more as-yet-unidentified inorganic superoxides or peroxides in the martian soil. Using electron paramagnetic resonance spectroscopy, we show that superoxide radical ions (O2-) form directly on Mars-analog mineral surfaces exposed to ultraviolet radiation under a simulated martian atmosphere. These oxygen radicals can explain the reactive nature of the soil and the apparent absence of organic material at the martian surface.