No abstract

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21 WAF1/Cip1 , p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/ Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.

It has been proposed that family VIII carboxylesterases and class C ␤-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze ␤-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various ␤-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C ␤-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze ␤-lactam antibiotics. EstU1 was able to hydrolyze firstgeneration ␤-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the ␤-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.

Many of biochemical or physiological processes can be regulated by non-coding RNAs as well as coding RNAs in plants, animals and microbes. Recently, many small RNAs including microRNAs (miRNAs) and endogenous small interference RNAs (siRNAs) and long non-coding RNAs have been discovered from ubiquitous organisms including plants. Biotic and abiotic stresses are main causal agents of crop losses all over the world. Much efforts have been performed for understanding the complex mechanism of stress responses. Up to date, many of these researches have been related with the identification and investigation of stress-related proteins, showing limitation to resolve the complex mechanism. Recently, noncoding RNAs as well as coding genes have been gradually interested because of its potential roles in plant stress responses as well as other biophysical aspects. In this review, various potential roles of non-coding RNAs, especially miRNAs and siRNAs, are reviewed in relation with plant biotic and abiotic stresses.

Efficient extraction of total proteins from soil microorganisms is tedious because of small quantity. In this regard, an improved method for extraction of whole cell proteins is developed from soil microorganisms, Saccharomyces cerevisiae and Pichia pastoris. of which the cell wall are very strong. Pretreatment with NH4OH prior to the final extraction using NaOH/ SDS was tried under the basis that ammonium ion was possible to enhance the permeability and/or to weaken the yeast cell walls. The pre-treatment of yeast cells with NH4OH drastically enhanced the protein extraction when it was compared with control (without NH4OH pre-treatment). At the pre-treatment of 0.04 N NH4OH at pH 9.0, about 3 fold of proteins was obtained from p. pastoris. Ammonium hydroxide appears to penetrate into the yeast cell walls more readily at basic pH. The effect of NH4OH pretreatment was pH dependent. The methods developed in this experiment might be applicable for an effective extraction of yeast proteins for the purpose of biochemical studies, especially proteomic analysis.