Chloramphenicol Acetyltransferase Expression Research Articles

The Pioneer platform: A novel approach for selection of selective anti-cancer cytotoxic activity in bacteria through co-culturing with engineered human cells.

Most of the small-molecule drugs approved for the treatment of cancer over the past 40 years are based on natural compounds. Bacteria provide an extensive reservoir for the development of further anti-cancer therapeutics to meet the challenges posed by the diversity of these malignant diseases. While identifying cytotoxic compounds is often easy, achieving selective targeting of cancer cells is challenging. Here we describe a novel experimental approach (the Pioneer platform) for the identification and development of 'pioneering' bacterial variants that either show or are conduced to exhibit selective contact-independent anti-cancer cytotoxic activities. We engineered human cancer cells to secrete Colicin M that repress the growth of the bacterium Escherichia coli, while immortalised non-transformed cells were engineered to express Chloramphenicol Acetyltransferase capable of relieving the bacteriostatic effect of Chloramphenicol. Through co-culturing of E. coli with these two engineered human cell lines, we show bacterial outgrowth of DH5α E. coli is constrained by the combination of negative and positive selection pressures. This result supports the potential for this approach to screen or adaptively evolve 'pioneering' bacterial variants that can selectively eliminate the cancer cell population. Overall, the Pioneer platform demonstrates potential utility for drug discovery through multi-partner experimental evolution.

Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium

Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.

Preventive Effect of Blueberry Extract on Liver Injury Induced by Carbon Tetrachloride in Mice.

The blueberry is a common fruit that is rich in nutritional value and polyphenol substances. In this study, the blueberry polyphenol content in extract was analysed by spectrophotometry. The results showed that the blueberry polyphenol content in the extract reached 52.7%. A mouse model of liver injury induced by carbon tetrachloride (CCl4) was established to study the preventive effect of blueberry extract (BE) on liver injury in mice and the experimental animals were examined using biochemical and molecular biological methods. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are important clinical liver function indicators; the changes of triglyceride (TG) and total cholesterol (TC) are observed after liver injury; interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are important inflammatory indexes; superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) are important changes of oxidative stress indexes. The in vivo animal experiment results showed that BE decreased the liver index of mice with liver injury, BE could reduce the AST, ALT, TG and TC levels and also could reduce the serum cytokine IL-6, TNF-α and IFN-γ levels in mice with liver injury. Moreover, BE increased the SOD activity and decreased the TBARS level in the gastric tissues of mice with liver injury. After treatment with the highest concentration of BP in liver injury mice, these levels returned close to those obtained after treatment with the standard drug of silymarin. Detection of messenger RNA (mRNA) in liver tissue showed that BE upregulated the Cu/Zn-SOD, Mn-SOD and chloramphenicol acetyltransferase (CAT) expression levels and downregulated cyclooxygenase (COX)-2 expression. The effect of BE on mice with liver injury was positively correlated with the BE concentration and was similar to that of silymarin, which is a drug for liver injury, suggesting that BE had a good preventive effect on liver injury. Thus, BE rich in polyphenols is a bioactive substance with value for development and utilization.

Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay

TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

Abstract 050: Endothelin-1 Overexpression In Endothelial Cells Increases Blood Pressure In An Endothelin Type A Receptor-Dependent Manner

Introduction: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells (EC) are complex and remain unclear. Transgenic mice with constitutive EC-specific human ET-1 ( EDN1 ) overexpression presented vascular injury but no change in BP, which could be due to adaptation to life-long high ET-1 exposure. We have now generated an inducible EC-restricted EDN1 overexpressing mouse (ieET-1) in order to demonstrate the effects of ET-1 on BP regulation independent of developmental effects. Method: Two transgenic mouse lines (C134 and C170) expressing chloramphenicol acetyltransferase ( cat ) and EDN1 before and after Cre-mediated excision, respectively, were crossed with mice expressing tamoxifen-inducible CreER T2 under the control of Tie2 promoter (ieCre) to generate ieET-1 mice. Mice were treated with tamoxifen (1 mg/kg/day, SC) or vehicle for 5 days and sacrificed 16 days later. Additional mice were treated with 5 or 10 mg/kg/day PO of ET type A receptor blocker, atrasentan, from day 10. BP was determined by telemetry. Plasma ET-1 levels were assessed by ELISA. ET type A and B receptors expression, Ednra and Ednrb , were evaluated in the kidney by quantitative PCR. Results: Tamoxifen increased plasma ET-1 in ieET-1 C134 and C170 (7.1±0.7 and 13.2±2 pg/mL, respectively, P <0.01), when compared to control ieET-1 C134 and C170 (0.7±0.2 and 1.0±0.3 pg/mL) and tamoxifen-treated ieCre (0.8±0.1 pg/mL). ET-1 overexpression increased night systolic BP in ieET-1 C134 and C170 (137±4 and 132±4 mmHg) compared to tamoxifen-treated ieCre (116±7 mmHg), which was reversed partially or completely with 5 or 10 mg/kg/day of atrasentan, respectively (122±3 and 115±4 mmHg, P <0.01). ET-1 overexpression in ieET-1 C134 was accompanied by increased Ednra expression in renal cortex and medulla (2.9±0.6 and 8.2±3.0 fold, P <0.05) and Ednrb levels in the renal cortex (2.0±0.3 fold, P <0.05) and a trend to increased levels in renal medulla (3.8±1.3 fold) compared to control ieET-1 C134. Conclusion: Our results demonstrate that this new inducible EC-restricted EDN1 overexpressing mouse exhibits ET-1-dependent elevated BP mediated by ET type A receptors. The increased expression of renal ET receptors could play a role in ET-1-induced BP rise.

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells.

The β-globin matrix attachment regions (MARs) were inserted into the 5'-site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number.

Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells

BackgroundThere are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5′ flanking region of the mouse cirp gene.ResultsBy transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5′ flanking region octanucleotide 5′-TCCCCGCC-3′ is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5′ flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5′ flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C.ConclusionsCold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker.

Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.

Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.

A new inducible expression system in a transformed green alga, Chlorella vulgaris

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.

Proteasomal Degradation Is Transcriptionally Controlled by TCF11 via an ERAD-Dependent Feedback Loop

Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.

HIV-1 Reactivation Induced by the Periodontal Pathogens Fusobacterium nucleatum and Porphyromonas gingivalis Involves Toll-Like Receptor 4 and 9 Activation in Monocytes/Macrophages

ABSTRACT Although oral coinfections (e.g., periodontal disease) are highly prevalent in human immunodeficiency virus type 1-positive (HIV-1 + ) patients and appear to positively correlate with viral load levels, the potential for oral bacteria to induce HIV-1 reactivation in latently infected cells has received little attention. We showed that HIV-1 long terminal repeat (LTR) promoter activation can be induced by periodontopathogens in monocytes/macrophages; nevertheless, the mechanisms involved in this response remain undetermined. Since Toll-like receptor 2 (TLR2), TLR4, and TLR9 activation have been involved in HIV-1 recrudescence, we sought to determine the role of these TLRs in HIV-1 reactivation induced by the periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis using BF24 monocytes/macrophages stably transfected with the HIV-1 promoter driving chloramphenicol acetyltransferase (CAT) expression and THP89GFP cells, a model of HIV-1 latency. We demonstrated that TLR9 activation by F. nucleatum and TLR2 activation by both bacteria appear to be involved in HIV-1 reactivation; however, TLR4 activation had no effect. Moreover, the autocrine activity of tumor necrosis factor alpha (TNF-α) but not interleukin-1β (IL-1β) produced in response to bacteria could impact viral reactivation. The transcription factors NF-κB and Sp1 appear to be positively regulating HIV-1 reactivation induced by these oral pathogens. These results suggest that oral Gram-negative bacteria ( F. nucleatum and P. gingivalis ) associated with oral and systemic chronic inflammatory disorders enhance HIV-1 reactivation in monocytes/macrophages through TLR2 and TLR9 activation in a mechanism that appears to be transcriptionally regulated. Increased bacterial growth and emergence of these bacteria or their products accompanying chronic oral inflammatory diseases could be risk modifiers for viral replication, systemic immune activation, and AIDS progression in HIV-1 + patients.

Supernatants from oral epithelial cells and gingival fibroblasts modulate Human Immunodeficiency Virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages

Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes/macrophages, albeit this effect is most notable following direct stimulation of the cells with oral gram-negative bacteria.

Glutathione synthesis inhibitor butathione sulfoximine regulates ceruloplasmin by dual but opposite mechanism: Implication in hepatic iron overload

Glutathione (GSH) depletion is often detected in chronic pathological conditions like hepatitis C infection, alcohol consumption or xenobiotic assault with simultaneous reactive oxygen species (ROS) generation and hepatic iron overload. However, relation between GSH depletion and regulators of iron homeostasis is not clear so far. To determine that hepatic HepG2 cells were treated with GSH synthesis inhibitor butathione sulfoximine (BSO) and a dual regulation of ceruloplasmin (Cp) that involves in hepatic iron release was detected unlike other iron homeostasis regulators. BSO treatment that caused marginal GSH deficiency increased Cp synthesis due to increased transcription mediated by activator protein (AP)-1-binding site. In higher GSH deficiency (> 40 %) with increased ROS generation, Cp expression was decreased due to promotion of Cp mRNA decay mediated by 3'untranslated region (3'UTR) as found by transfecting chimera of chloramphenicol acetyl transferase (CAT) gene with Cp 3'UTR. RNA gel shift assay showed significant reduction in 3'UTR binding protein complex in similar condition. Decreased CAT expression and RNA-protein complex binding are reversed by pretreatment with antioxidant N-acetyl cysteine suggesting 3'UTR binding protein complex is redox-sensitive. This unique and opposite regulation of Cp provides a mechanism of hepatic iron-deposition during glutathione deficiency detected in chronic pathological conditions.

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese-hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human beta-globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 59 or 39 site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 59 site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 39 site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.

Biological Pacemaker Engineered by Nonviral Gene Transfer in a Mouse Model of Complete Atrioventricular Block

We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

The C, V and W proteins of Nipah virus inhibit minigenome replication

Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.

Allosteric Inhibition of the HIV-1 Rev/RRE Interaction by a 3′-Methylphosphonate Modified Antisense Oligo-2′-O-Methylribonucleotide

The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stemloops, is found in all spliced and partially spliced human immunodeficiency virus (HIV) mRNA transcripts. The RRE interacts with HIV-encoded Rev protein, which facilitates exit of the transcripts from the nucleus to the cytoplasm. Because the Rev/RRE interaction is critical to virus function, it is considered a potential target for therapeutic drugs. We have investigated the interactions of antisense oligonucleotides with stem-loop II, a region that contains the high-affinity binding site for Rev. Oligo-2'-O-methylribonucleotides terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were targeted to the 5'- or 3'-side of stem-loop IIB, which is adjacent to the Rev binding site. Thermal denaturation experiments showed that oligonucleotides of this type form highly stable duplexes with complementary single-stranded RNA. Gel electrophoretic mobility shift assays (EMSA) showed that the oligonucleotides bound with high affinity and specificity at 37 degrees C to RRE stem-loop II RNA with apparent dissociation constants, K(D), in the low nM range. A 16-mer, 2-1mp, whose K(D) is 46 nM, competitively inhibited binding of Rev peptide to RRE stem-loop II RNA as shown by EMSA experiments. When transfected into HEK 293T cells, 2-1mp inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) by 60% at a concentration of 300 nM oligonucleotide. These results are consistent with a mechanism by which 2-1mp blocks access of Rev to the RRE/CAT transcript thus preventing nuclear export and subsequent translation.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.