Functional characterization of the human placental fusogenic membrane protein syncytin 2

Fusion of cytotrophoblasts into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. The envelope protein of a human endogenous retrovirus W (HERV-W) family member, syncytin 1, has been shown to mediate placental cell fusion. Recently, the envelope protein of another HERV family member (HERV-FRD), syncytin 2, has been identified and shown to be highly expressed in the placenta. To better understand the biology of syncytin 2, in this study we first investigated syncytin 2 gene expression in normal and preeclamptic placentas and then characterized the functions of syncytin 2. The expression of syncytin 2 gene was decreased in preeclamptic placentas and could be stimulated by the cAMP stimulant forskolin. The endoprotease furin was found to be involved in the posttranslational cleavage of syncytin 1 and 2 polypeptides into surface and transmembrane subunits. In addition, proper association of the subunits of syncytins 1 and 2 is probably required for the functional integrity of each protein, because subunit swapping of syncytins 1 and 2 failed to generate fusogenic chimeras. Finally, we demonstrated that the disulfide bridge-forming CX(2)C and CX(7)C motifs found in syncytins 1 and 2 are essential for their fusogenic activities, because mutations in the CX(2)C motif not only abolished fusogenesis but also functioned as dominant-negative mutants. Our results suggest that syncytin 2 may function as a second fusogenic protein for placental cell fusion.     

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

Background  

Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others.     

Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells

Background  

Porphyromonas gingivalis in subgingival dental plaque, as part of a mature biofilm, has been strongly implicated in the onset and progression of chronic periodontitis. In this study using DNA microarray we compared the global gene expression of a P. gingivalis biofilm with that of its planktonic counterpart grown in the same continuous culture.     

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO‐111 against blister blight disease of tea

Aims

The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition.

Methods and Results

A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO‐075, BMO‐111 and BMO‐147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36‐h‐old culture of BMO‐111 (1 × 10⁸ colony‐forming units ml^?1) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO‐111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO‐111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses.

Conclusions

It could be concluded that the biocontrol agent O. anthropi BMO‐111 was effective against blister blight disease of tea.

Significance and Impact of the Study

Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.     

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

Background  

The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.     

Growth of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in a long spiral form does not alter expression of immunodominant proteins

Background  

We have previously reported that altered culture conditions (a broth media with shaking) could induce a strain of Helicobacter pylori to assume a long spiral morphology resembling that described for Helicobacter heilmannii. The present study was initiated to determine if other strains of H. pylori could be induced to assume that morphology and if doing so would alter the expression of immunodominant proteins.     

Monitoring <Emphasis Type="Italic">ALS1</Emphasis> and <Emphasis Type="Italic">ALS3</Emphasis> Gene Expression During In Vitro <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation Under Continuous Flow Conditions

ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear.     

Syncytins - retroviral envelope genes captured for the benefit of placental development

In the past, germline infections by retroviruses have led to vertical transmission of "endogenized" retroviruses. Escaping genetic drift, some of the viral genes have been conserved until now because of beneficial effects on their host. Here we present the syncytin genes that encode envelope proteins from endogenous retroviruses. Syncytins have inherited fusogenic properties from their infectious ancestor and are specifically expressed in the placenta. Both properties have suggested their involvement in the formation of the syncytiotrophoblast, a multinucleated layer that mediates feto-maternal exchanges in the placenta. The capture of syncytin genes occurred on several independent occasions during evolution of mammals. Knock-out experiments of syncytins in the mouse definitively confirmed the role of these genes in placentation. Finally, a second function for syncytins, i.e. an immunosuppressive activity, could contribute to materno-fetal immune tolerance. This constitutes a remarkable example of convergent evolution where the properties of retroviral envelope genes are subverted to play a major physiological role.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

Optimization of the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> nisin-controlled gene expression system NICE for industrial applications

Background  

The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein.     

Physiological responses to folate overproduction in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WCFS1

Background  

Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

Troglitazone, a PPAR-γ activator prevents endothelial cell adhesion molecule expression and lymphocyte adhesion mediated by TNF-α

Background  

Cytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1) expression is associated with the onset and progression of inflammatory bowel disease (IBD).     

In vitro culture and somatic cell nuclear transfer affect imprinting of SNRPN gene in pre- and post-implantation stages of development in cattle

Background  

Embryo in vitro manipulations during early development are thought to increase mortality by altering the epigenetic regulation of some imprinted genes. Using a bovine interspecies model with a single nucleotide polymorphism, we assessed the imprinting status of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene in bovine embryos produced by artificial insemination (AI), in vitro culture (IVF) and somatic cell nuclear transfer (SCNT) and correlated allelic expression with the DNA methylation patterns of a differentially methylated region (DMR) located on the SNRPN promoter.     

Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration

Introduction  

The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.     

Effect of codon optimization and subcellular targeting on <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice

Background  

Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG1_78-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

Improved mycobacterial protein production using a <Emphasis Type="Italic">Mycobacterium smegmatis groEL1ΔC</Emphasis>expression strain

Background  

The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1.     

Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties

Aim

To evaluate the suitability of marine lactic acid bacteria (LAB) as starter cultures for Sargassum sp. fermentation to enhance its antioxidant and anticoagulation activity.

Methods and Results

LAB isolated from marine source were characterized for their ability to utilize seaweed as a sole carbon source and applied to Sargassum fermentation. Fermentation period was optimized by monitoring the fermented sample at regular interval for a period of 18 days. Results revealed that a fermentation period of 12 days was effective with maximum culture viability and other desirable characteristics such as pH, total titratable acidity, total and reducing sugars. Under optimum fermentation period, the sample fermented with P1‐2CB‐w1 (Enterococcus faecium) exhibited maximum anticoagulation activity and antioxidant activity.

Conclusions

The study reveals a novel well‐defined starter culture from marine origin intended for seaweed fermentation for recovery of bioactive molecules.

Significance and Impact of the study

The study provides information for the enhancement of bioactive molecules in an eco‐friendly manner and also paves a way towards the development of wide range of seaweed functional foods.