Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1 mM sulindac over 16 h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1 mM sulindac treatment for 8 h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30 K, 3 K and 1 K). Proteins isolated in the > 30 K and 3–30 K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3 K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1 mM sulindac for 8 h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5 AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8 h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1 mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture

Cocultures bear great potential in the conversion of complex substrates and process intensification, as well as, in the formation of unique components only available due to inter-species interactions. Dynamic data of coculture composition is necessary for understanding and optimizing coculture systems. However, most standard online determined parameters measure the sum of all species in the reactor system. The kinetic behavior of the individual species remains unknown. Up to now, different offline methods are available to determine the culture composition, as well as the online measurement of fluorescence of genetically modified organisms. To avoid any genetic modification, a noninvasive online monitoring tool based on the scattered light spectrum was developed for microtiter plate cultivations. To demonstrate the potential, a coculture consisting of the bacterium Lactococcus lactis and the yeast Kluyveromyces marxianus was cultivated. Via partial least squares regression of scattered light spectra, the online determination of the individual biomass concentrations without further sampling and analyses is possible. The results were successfully validated by a Coulter counter-analysis, taking advantage of the different cell sizes of both organisms. The findings prove the applicability of the new method to follow in detail the dynamics of a coculture.     

Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six β-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L⁻¹. The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L⁻¹ (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.     

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.     

暗黑菌(Atribacteria)的环境分布和功能特征

暗黑菌门包括OP9和JS1两大分支,成员大多为未培养微生物,在自然环境中广泛分布,并且在部分环境如厌氧海洋沉积物、地热环境以及油藏中为优势微生物。基于基因组信息的研究表明,暗黑菌为严格的厌氧微生物,同时具有降解糖类、小分子酸、短链正构烷烃的能力,在地球碳循环过程中可能扮演着重要的角色。然而,由于缺乏相应代表性的纯培养菌株,对于暗黑菌的生理生化功能推测仍有待验证。文章概述了暗黑菌的发现及发展历史,分析了其环境分布和多样性,简述了目前提出的三种代谢方式,提出了未来暗黑菌的研究发展方向。     

Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.     

Genetic and physiological relatedness of antagonistic Trichoderma isolates against soil borne plant pathogenic fungi

In this study, the in vitro potential of 42 Trichoderma spp. were evaluated against four isolates of soil borne phytopathogenic fungi viz., Rhizoctonia solani, Macrophomina sp., Sclerotium rolfsii and Pythium aphanidermatum in dual culture techniques and through production of volatile and non-volatile inhibitors. In vitro screening results showed that the proportion of isolates with antagonistic activities was highest for the S. rolfsii followed by R. solani, Macrophomina sp. and P. aphanidermatum, respectively. The isolates TNT1, TNP2 and TWP1 showed consistent results in volatile and non-volatile activity in vitro against any of the two pathogens tested. Based on genomic finger prints, potential isolates showed no particular correlation between the origin of the isolates and the Random Amplified Polymorphic DNA (RAPD) groups could not be established. However, the polymorphism shown by the isolates did not correlate to their level of antagonism. Whereas, in physiology studies using BIOLOG (microbial identification system), three groups were formed, one group consists with 14 different Trichoderma species and two groups with two isolates each comprised of only T. koningii and T. viride.