Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Differential expression of CD45 on canine microglial cells

CD45, also called leucocyte common antigen is a transmembrane protein tyrosine phosphatase on the surface of nearly all white blood cells and has a functional role in signal transduction. In the brain, the expression of CD45 can be used to distinguish microglial cells with a characteristic phenotype of CD11b/c+ and CD45(low) from other central nervous system (CNS) macrophages which show an expression of CD11b/c+ and CD45(high). In the course of pathological changes in the CNS, microglia in rodents is known to readily upregulate expression of various surface molecules, such as CD45. Understanding the mechanisms that regulate expression of surface molecules is essential to study the pathogenesis of CNS diseases. In the present study, the expression of CD45 on microglia of 42 dogs was examined ex vivo by means of flow cytometry. The dogs were classified in two groups according to the histopathological diagnosis in the CNS. All dogs without changes in the CNS (group I; n = 22) only showed low percentages of CD45+ microglial cells. In group II consisting of 20 dogs with different intracranial diseases varying results were obtained. Thirteen dogs showed a low percentage of CD45+ microglial cells whereas seven dogs exhibited high percentages of microglial cells expressing CD45. Evaluation of expression intensity in these seven dogs revealed two subpopulations of CD45+ microglial cells: a large subpopulation with CD45(low) and a small subpopulation with CD45(high). The expression intensity of CD45(high) was comparable with that of canine monocytes. It was attempted to correlate these findings to age of the animals, underlying disease, duration of clinical signs, medical treatment, occurrence of seizure activity and the expression of other surface molecules. It appeared that dogs with high percentages of CD45+ suffered from long-lasting CNS disease with seizures. In future studies, the reason and consequences for upregulated CD45 in long-lasting CNS diseases has to be further evaluated.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.

GPR87 is an overexpressed G-protein coupled receptor in squamous cell carcinoma of the lung

Lung cancer is the leading cause of cancer death worldwide. The overall 5-year survival after therapy is about 16% and there is a clear need for better treatment options, such as therapies targeting specific molecular structures. G-protein coupled receptors (GPCRs), as the largest family of cell surface receptors, represent an important group of potential targets for diagnostics and therapy. We therefore used laser capture microdissection and GPCR-focused Affymetrix microarrays to examine the expression of 929 GPCR transcripts in tissue samples of 10 patients with squamous cell carcinoma and 7 with adenocarcinoma in order to identify novel targets in non-small cell lung carcinoma (NSCLC). The relative gene expression levels were calculated in tumour samples compared to samples of the neighbouring alveolar tissue in every patient. Based on this unique study design, we identified 5 significantly overexpressed GPCRs in squamous cell carcinoma, in the following decreasing order of expression: GPR87 > CMKOR1 > FZD10 > LGR4 > P2RY11. All are non-olfactory and GRAFS (glutamate, rhodopsin, adhesion, frizzled/taste2, secretin family) classified. GPR87, LGR4 and CMKOR1 are orphan receptors. GPR87 stands out as a candidate for further target validation due to its marked overexpression and correlation on a mutation-based level to squamous cell carcinoma.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation

Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens

The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Cultivation strategies to enhance productivity of Pichia pastoris: A review.

Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

The liver stage of Plasmodium berghei inhibits host cell apoptosis.

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite-host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei-infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei-infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-alpha/D-galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-alpha-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.

Transcriptional response to cardiac injury in the zebrafish: systematic identification of genes with highly concordant activity across in vivo models

BACKGROUND Zebrafish is a clinically-relevant model of heart regeneration. Unlike mammals, it has a remarkable heart repair capacity after injury, and promises novel translational applications. Amputation and cryoinjury models are key research tools for understanding injury response and regeneration in vivo. An understanding of the transcriptional responses following injury is needed to identify key players of heart tissue repair, as well as potential targets for boosting this property in humans. RESULTS We investigated amputation and cryoinjury in vivo models of heart damage in the zebrafish through unbiased, integrative analyses of independent molecular datasets. To detect genes with potential biological roles, we derived computational prediction models with microarray data from heart amputation experiments. We focused on a top-ranked set of genes highly activated in the early post-injury stage, whose activity was further verified in independent microarray datasets. Next, we performed independent validations of expression responses with qPCR in a cryoinjury model. Across in vivo models, the top candidates showed highly concordant responses at 1 and 3 days post-injury, which highlights the predictive power of our analysis strategies and the possible biological relevance of these genes. Top candidates are significantly involved in cell fate specification and differentiation, and include heart failure markers such as periostin, as well as potential new targets for heart regeneration. For example, ptgis and ca2 were overexpressed, while usp2a, a regulator of the p53 pathway, was down-regulated in our in vivo models. Interestingly, a high activity of ptgis and ca2 has been previously observed in failing hearts from rats and humans. CONCLUSIONS We identified genes with potential critical roles in the response to cardiac damage in the zebrafish. Their transcriptional activities are reproducible in different in vivo models of cardiac injury.

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.