Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp.

AIMS: The aim of this study was to develop a real-time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). METHODS AND RESULTS: A duplex real-time PCR specific for the genes encoding for ß-glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety-six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. CONCLUSIONS: The duplex real-time PCR assay was found to be simple, rapid, reliable and specific. SIGNIFICANCE AND IMPACT OF THE STUDY: If possible at all, delineation of so-called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno- and serotypes. This assay clearly simplifies the differentiation of both.

Cross-talk between signal transducer and activator of transcription (Stat5) and thyroid hormone receptor-beta 1 (TRbeta1) signaling pathways.

PRL and T3 are involved in antagonistic regulations during various developmental processes in vertebrate species. We have studied cross-talk between transcription factors activated by these signaling pathways, i.e. signal transducer and activator of transcription 5 (Stat5) and thyroid hormone receptor beta1 (TRbeta1). Liganded TRbeta1 in the presence of its heterodimeric partner, retinoid X receptor gamma (RXRgamma), inhibited the PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60%. This T3-inhibitory effect studied on Stat5 activity was partly reversed by overexpression of a TRbeta1 dominant negative variant mutated within its nuclear localization signal (TR2A). We next showed that TRbeta1 and TR2A in the presence of RXRgamma increased and decreased, respectively, Stat5 localization into the nucleus regardless of hormonal stimulation. Thus, our data suggest that TRbeta1 can be associated with Stat5 in the cytoplasm and may be involved in Stat5 nuclear translocation. In PRL-treated cells overexpressing TRbeta1/RXRgamma, both Stat5 and TRbeta1 were coimmunoprecipitated, indicating physical association of the two transcription factors. In these cells, addition of T3 with ovine (o)PRL decreased the amounts of total and tyrosine-phosphorylated Stat5 in the cytoplasm compared with oPRL-treated cells. In the nucleus, no clear difference was observed on Stat5 DNA-binding after treatment with PRL and T3 vs. PRL alone in TRbeta1/RXRgamma transfected cells. However, antibodies directed against TRbeta1 lowered Stat5-DNA binding and addition of the deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5 transcriptional activity. Thus, we postulated that the negative cross-talk between TR and Stat5 on target genes could involve histone deacetylase recruitment by liganded TRbeta1.

T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer.

The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.

Use of heterologous DNA-based gene transfer to follow physiological, T3-dependent regulation of myosin heavy chain genes in Xenopus tadpoles.

In vivo gene transfer and RNase protection assay were used to follow thyroid hormone (T3)-dependent regulation of myosin heavy chain (myHC) genes in Xenopus tadpole dorsal muscle. One embryonic and one adult myHC form were measured by each approach. RNase protection assay showed that T3 decreased expression of endogenous embryonic mRNA (E3), but increased adult (A7) transcripts. Gene transfer showed that T3 exerted transcriptional effects on mammalian embryonic and adult myHc promoters injected into the same muscle. The kinetics and profiles of the transcriptional responses were superimposable on endogenous responses. The results strengthen the use of in vivo approaches for determining the roles of transcription factors and cis-regulatory sequences in integrated contexts.

Activation of gene transcription by tilapia prolactin variants tiPRL188 and tiPRL177.

In the tilapia species Oreochromis niloticus, the pituitary releases two forms of prolactins (tiPRL188 and tiPRL177). The binding parameters and the activation of tiPRL-induced JAK2/Stat5 signalling pathway were analysed using a mammalian cell line transiently transfected with the tiPRL receptor (tiPRLR). Our data indicate that the tiPRLR is able to mediate transcriptional activation of the PRL responsive element. At nanomolar concentrations, tiPRL188 activates gene transcription whereas at micromolar concentrations it inhibits luciferase transcription from the lactogenic responsive element. This is consistent with a model of receptor dimerisation. In contrast, the activation by tiPRL177 was only reached at high (microM) concentrations. The transcriptional activities induced by tiPRL177 and tiPRL188 are discussed in the context of the physiology of these hormones.

Tail regression, apoptosis and thyroid hormone regulation of myosin heavy chain isoforms in Xenopus tadpoles.

T3 effects on myosin heavy chain gene expression were analysed in muscles undergoing different fates during metamorphosis. Muscle fate was followed by somatic gene transfer of a constitutively expressed luciferase vector. Persistent expression was found in dorsal muscle which is remodelled during metamorphosis whilst the signal disappeared in apoptosing caudal muscle. RNAse protection assay was used to follow production of myosin heavy chain isoforms: two isoforms identified as embryonic (E3 and E19) and one adult form (A7). The effects of T3 treatment were followed over 120 h. During this time frame E3 and A7 expression patterns were found to be similar in both caudal and dorsal muscles. Most notably, at 48 h E3 expression was significantly down-regulated and production of A7 significantly upregulated in both caudal and dorsal muscle. Thus T3-induced transitions in muscle gene expression are independent of muscle fate during amphibian metamorphosis.

[Modulation of T4 to T3 conversion. Comparative aspects (author's transl)]. / Modulation de la conversion de T4 en T3. Aspects comparatifs.

The peripheral conversion of T4 to T3 has been demonstrated in primitive Vertebrates, larval Lampreys (which do not have thyroid follicles but synthesize T3 and T4 in the endostyle) and in more evolved Vertebrates. Up to now the presence of rT3 has never been demonstrated in fish, its eventual role in Amphibia remains to be established but seems important in chick embryo. The conversion of T4 to T3 is stimulated during amphibian metamorphosis and in salt water fish, concurrently with stimulation of thyroidal secretion. In fish, prolactin stimulates 5-deiodination of T4 directly without the involvement of the pituitary thyroid axis.

[Stimulation of peripheral conversion of thyroxine to triiodothyronine by ovine somatotropic hormone in the eel (Anguilla anguilla L.)]. / Stimulation de la conversion périphérique de la thyroxine en triiodothyronine par l'hormone somatotrope ovine chez l'anguille (Anguilla anguilla L.).

One injection of ovine growth hormone (1.27 X 10(-5) USP units/g body weight) determines in the Eel, 1 day later, a twofold decrease in plasma thyroxine level as well as a twofold increase in plasma triiodothyronine level. These modifications of circulating iodothyronine levels result from a considerable stimulation of the peripheral deiodination of thyroxine to triiodothyronine, shown by a kinetic study of labelled thyroxine metabolism. The increase of production of metabolically active hormone, triiodothyronine, induced by growth hormone could represent the (or one of the) mechanism(s) implicated in the synergic action of these two hormones on Vertebrate growth.

Fish growth hormone enhances peripheral conversion of thyroxine to triiodothyronine in the eel (Anguilla anguilla L.).

In the eel, a low dose of tilapia growth hormone (tGH) (45 ng/g body wt), like ovine GH (oGH), induces a decrease in plasma thyroxine and a concomitant increase in plasma triiodothyronine, which result from a stimulation of peripheral conversion of thyroxine to triiodothyronine. Salmon prolactin (sPrl), unlike ovine Prl (oPrl), has no such action. Recognition of this specific action of growth hormone (GH) on production of active thyroid hormone (T3) opens up a new approach to the problem of the action of both hormones (GH, T3) in growth and in seawater adaptation of fish.

Binding and degradation of 3,5,3'-triiodothyronine and thyroxine by rat intestinal bacteria.

Intestinal bacteria hydrolyze conjugates of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) secreted in bile, but it is not clear whether they have any other role in metabolism, storage, transport, or action of thyroid hormone in the intestines. We have examined aspects of T3 and T4 binding and degradation processes in fresh feces and cecum contents, obtained from normal control rats and from rats partially decontaminated by treatment with oral antibiotics for 2-3 wk. Samples were homogenized in phosphate buffer, fractionated, and subjected to various test conditions and incubated at 37 degrees C with 125I-labeled T3 (T3*) or T4 (T4*) for 2 or 24 h. Supernatants of high-speed centrifuged incubates were chromatographed to test for degradation products, and percentage binding was measured in the pellets. Substantial binding of T3* and T4* was found in all control rat feces and cecum content samples by 2 h, but binding was absent or significantly reduced in partially decontaminated rat samples. Bacterial binding of T3* and T4* were further shown to be competitive with graded doses of bovine serum albumin. Considerable degradation of T3* and T4* to labeled iodide (I*) only was also observed in feces and cecum content samples and was much greater in control rat than in corresponding partially decontaminated rat samples. Light had no effects in our system and heat reduced I* production. Propylthiouracil and sodium ipodate had little effect or equivocal effects, but dithiothreitol substantially inhibited I* production.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone-dependent transcriptional regulation of exogenous genes transferred into Xenopus tadpole muscle in vivo.

Metamorphosis in amphibians is marked by dramatic thyroid hormone-induced changes that include tail regression. To examine thyroid hormone effects on gene transcription during the early stages of tail resorption, we injected exogenous genes directly into the caudal skeletal muscle of Xenopus tadpoles and followed their expression in vivo. Gene expression was both strong and reproducible, and it correlated with the amount of DNA injected. Moreover, expression continued as long as the animals were blocked in prometamorphosis by antithyroid drugs (for up to 4 months). Thyroid hormone-dependent effects on transcription were examined by using a palindromic thyroid hormone response element linked to a chloramphenicol acetyltransferase reporter gene. Reporter gene expressions were normalized for transfection efficiency by using a constitutively expressed luciferase construct. Physiological concentrations of 3,5,3' triiodo-L-thyronine (1 nM), applied for 120 hr, produced a 5-fold increase in transcription (P < 0.05) from the thyroid hormone response element but did not modify transcription from constitutive viral promoters. This study thus demonstrates that by directly expressing genes in Xenopus tadpole muscle in vivo, one can exploit the powerful experimental advantages of gene transfer systems in an intact, physiologically normal animal.

Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.

Effects of vertebrate prolactins and growth hormones on thyroxine 5'-monodeiodination in the eel (Anguilla anguilla): a potential bioassay for growth hormone.

Growth hormones (GHs) and prolactins (Prls) purified from representatives of each vertebrate class from bony fish onwards were tested for their ability to stimulate in vivo peripheral deiodination of labeled thyroxine (T4*) into triiodo-L-thyronine (T3*) in the eel. Plasma T3*/T4* ratio was used as parameter. All GHs significantly increased T3*/T4*, the magnitude of the response being unrelated to the phylogenic position of species. No significant stimulation was shown with the various Prl, with the exception of ovine Prl, suggesting a heterosomatotropic effect of this preparation in the eel. Furthermore, both tilapia and ovine GH produced a dose-related effect on plasma T3*, T4*, and T3*/T4*. The stimulation of the peripheral deiodination of T4* into T3* estimated in vivo in the eel could become a specific, sensitive, and rapid fish bioassay for GH.

Apoptosis in Xenopus tadpole tail muscles involves Bax-dependent pathways.

Apoptosis is a fundamental mechanism implicated in normal development. One of the most spectacular developmental events involving apoptosis is tail regression during amphibian metamorphosis. We analyzed how thyroid hormone (3, 5, 3'-triiodothyronine, T3), the orchestrator of metamorphosis, affects expression and function of the proapoptotic gene Bax in the tail muscle of free-living Xenopus tadpoles. During natural metamorphosis Bax mRNA was expressed in tail muscles and was spatially correlated with apoptosis. Precocious treatment of tadpoles with T3 induced Bax expression and apoptosis. To verify that Bax expression was causally related to apoptosis we used a naked DNA gene transfer method to express Bax in the dorsal tail muscle. This induced apoptosis, and the process was exacerbated by T3 treatment. To determine whether T3 effects on Bax expression involved transcriptional regulation, we injected a Bax promoter sequence into dorsal and caudal tail muscles. In the dorsal muscle, T3 treatment did not affect transcription from the Bax promoter. However, in the caudal muscle, T3 treatment significantly increased Bax transcription. We conclude that T3-induced apoptosis in Xenopus tadpole tail muscles involves Bax-activating and Bax-synergis tic mechanisms. These programs are induced in spatially and temporally distinct manners.

Identification and modulation of a growth hormone-binding protein in rainbow trout (Oncorhynchus mykiss) plasma during seawater adaptation.

A soluble protein that specifically bound 125I-human growth hormone (hGH) was identified in rainbow trout plasma, using HPLC-gel filtration. The binding affinity of the protein for hGH was 1.2 x 10(9)M-1. 125I-rainbow trout GH (tGH) was also able to bind to the protein albeit with a lower affinity (6.6 x 10(7)M-1) than hGH. Crosslinking experiments using 125I-hGH revealed two specific bands of 150 and 130 kDa. The complex 125I-hGH-BP could be precipitated by a monoclonal anti-GH receptor antibody, suggesting a close relationship between the plasma GH-BP and the GH receptor. A fourfold increase in the hGH binding to the GH-BP was shown 48 h after transfer of the fishes from freshwater to seawater. The increase in binding was related to a high binding capacity without significant changes in binding affinity. These results suggest a potential role of this related GH-BP as an index of GH effects during seawater adaptation in salmonids.