LacZ Activity Research Articles

GyrA Interacts with MarR To Reduce Repression of themarRABOperon inEscherichia coli

Bacterial two-hybrid studies of randomly cloned Escherichia coli DNA identified a physical interaction between GyrA, subunit A of gyrase, and MarR, a repressor of the marRAB operon. GyrA-His immobilized on Ni-nitrilotriacetic acid (NiNTA) resin bound MarR, while MarR alone did not bind. GyrA interfered with MarR binding to marO, as detected by electrophoretic mobility assays. In a strain bearing the marRAB operon and a marO-lacZ reporter, overexpression of GyrA increased LacZ activity, indicating decreased repression of marO-lacZ by MarR. These results were confirmed by an increased survival of cells treated with quinolones and other antibiotics when GyrA was overexpressed. This work, like a previous study examining TktA (12), shows that unrelated proteins can regulate MarR activity. The findings reveal an unexpected regulatory function of GyrA in antibiotic resistance.

Designed Recombinant Transcription Factor with Antibody-Variable Regions

Many recombinant transcription factors have been invented, but we cannot select a substance used as an inducer. In this study, we have created a novel expression control system in which we can select a substance as an inducer toward which a monoclonal antibody (mAb) is prepared. The variable region fragments (Fvs) of the heavy and light chains (V(H) and V(L)) of the bisphenol A (BPA)-specific mAb BBA-2187 were each fused to the DNA-binding domain (DBD) of LexA and the transactivation domain (AD) of VP16. The association between the two recombinant proteins in the presence of BPA constituted a functional transcription factor. The recombinant proteins in which the DBD was fused to the N-terminal side of the Fv and in which the nuclear localization signal (NLS) was fused to the N-termini of the construct including the AD highly induced beta-galactosidase (lacZ) expression in recombinant yeast cells grown with BPA. When the Fvs of the polychlorinated biphenyl (PCB)-specific mAb 4444 were used, DBD-NLS-V(H) and NLS-AD-V(L) showed significantly increased lacZ activity in response to a PCB derivative. The Fv transcription factor may be useful in many fields such as gene therapeutics.

Topological analysis of a haloacid permease of a Burkholderi a sp. bacterium with a PhoA-LacZ reporter

Background2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system.ResultsThirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features.ConclusionThe PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.

The regulatory elements of araBAD operon, contrary to lac‐based expression systems, afford hypersynthesis of murine, and human interferons in Escherichia coli

The overexpression of four different interferons, i.e., murine interferon alpha1 and human interferons alpha1, alpha 8, and alpha 21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon alpha1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining beta-galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis.

In Vivo Optical Imaging of LacZ Expression Using lacZ Transgenic Mice

beta-Galactosidase (beta-gal) (encoded by the lacZ gene) has been widely used as a transgene reporter enzyme. The ability to image lacZ expression in living transgenic animals would further extend the use of this reporter. It has been reported that 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-d-galactopyranoside (DDAOG), a conjugate of beta-galactose and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one), is not only a chromogenic lacZ substrate but that the cleavage product has far-red fluorescence properties detectable by in vivo imaging. In an attempt to noninvasively image lacZ expression in vivo, we applied fluorescence imaging to a G protein-coupled receptor (GPR56), knockout (KO) mouse model, in which the lacZ gene is introduced in the GPR56 locus. Compared to wild-type (WT) mice, GPR56KO/LacZ mice showed three- to fourfold higher fluorescence intensity in the head area 5 min after tail-vein injection of DDAOG. beta-Gal staining in sections of whole brain showed strong lacZ expression in homozygotes, but not in WT mice, consistent with lacZ activity detected by in vivo imaging. The kidneys were also visualized with fluorescence imaging both in vivo and ex vivo, consistent with beta-gal staining findings. Our results demonstrate that fluorescence imaging can be used for in vivo real-time detection of lacZ activity by fluorescence imaging in lacZ transgenic mice. Thus, this technology can potentially be used to noninvasively image changes of certain endogenous molecules and/or molecular pathways in transgenic animals.

The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis

The riboflavin (vitamin B2) biosynthetic genes in Bacillus subtilis are transcribed simultaneously from the riboflavin promoter (Prib). The 5’-end of the nascent rib-mRNA carries a flavin mononucleotide (FMN) binding riboswitch, which regulates gene expression. The antibiotic roseoflavin from Streptomyces davawensis is a naturally occurring riboflavin analog, its mechanism of action is largely unknown. A recombinant B. subtilis strain carrying a copy of Prib-RFN fused to a promoterless lacZ reporter gene in the chromosomal amyE locus was grown in a minimal medium. Upon addition of roseoflavin to the growth medium the apparent LacZ activity in this strain was not significantly reduced. Similar experiments carried out on recombinant B. subtilis strains oversynthesizing the flavin transporters RibU (B. subtilis) or RibM (S. davawensis) produced still other results. In these strains, roseoflavin (as well as riboflavin) repressed LacZ synthesis indicating that the RFN riboswitch is a target for roseoflavin (or roseoflavin mononucleotide), which may at least in part explain its antibiotic activity.

A Novel Method for Targeted Gene Therapy in Ischemic Tissues through Viral Transfection of an Expression Cassette Containing Multiple Repetitions of Hypoxia Response Element

Increased levels of the transcription factor hypoxia inducible factor (HIF)-1 occur only in hypoxic tissue. The authors propose a therapeutic strategy that relies on HIF-1, the enhancer hypoxia response element (HRE), and the delivery vector adeno-associated virus-2 (AAV2) to direct ischemia specific gene therapy to skin. An expression cassette containing the CMV promoter driving the reporter gene green fluorescent protein (GFP) was used to assess cutaneous tropism of AAV2. Transfection of dermal fibroblasts and immortalized keratinocytes (HaCat) was assessed with flow cytometry. Human embryonic kidney 293 (HEK) cells were used to produce vector stocks and test the authors' therapeutic strategy in quadruplicate. An expression cassette with nine repeats of HRE linked to beta-galactosidase (LacZ) within the AAV2 vector was constructed. HEK cells were transfected and exposed to normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. LacZ activity was measured by conversion of galactoside red-beta-D-galactopyranoside. Approximately 50 percent of dermal fibroblasts and HaCat cells were transfected when treated with 1 x 10(4) genome copies/cell of AAV2-CMV-GFP. Using the same titration of AAV2-9HRE-LacZ, transfected HEK cells demonstrated LacZ activity of 0.496 +/- 0.068 U/microg in normoxia and 2.9 +/- 0.58 U/microg in hypoxia. Transfected cells exposed to 24 hours of hypoxia show greater than an 11-fold increase in LacZ activity (p < 0.05) compared with baseline normoxic controls. The authors' results confirm that AAV2 has in vitro tropism for skin-derived cell lines. Furthermore, HRE will drive gene expression in ischemia but not normoxia. This is the first step toward the authors' goal of HIF-1-regulated gene therapy to prevent ischemia related skin injury.

Inactivation of FIP200 Leads to Inflammatory Skin Disorder, but Not Tumorigenesis, in Conditional Knock-out Mouse Models

FIP200 (focal adhesion kinase family interacting protein of 200 kDa) has been shown to interact with other proteins to regulate several intracellular signaling pathways. To study a potential role of FIP200 in tumorigenesis and possibly other disease processes in vivo, we created and analyzed murine mammary tumor virus-Cre-mediated FIP200 conditional knock-out (CKO) mice. We found that deletion of FIP200 in mammary epithelial cells did not result in spontaneous development of breast cancer. Moreover, deletion of FIP200 did not further accelerate or inhibit lymphomagenesis induced by inactivation of p53 in mice. Interestingly, however, FIP200 and p53 double conditional knock-out (dCKO) mice exhibited significant hyperplasia of epidermis (acanthosis), thickening of the cornified layer (hyperkeratosis), and increased vascularity in the dermis. FIP200 CKO mice also showed similar, although less severe, skin defects as dCKO mice. Analyses of primary keratinocytes isolated from dCKO mice did not detect increased proliferation of these cells in vitro, suggesting that epidermis hyperproliferation is not epidermal cell-autonomous but may be a consequence of increased inflammation triggered by immune cells in vivo. Consistent with this possibility, we found infiltration of leukocytes including T cells, macrophages, and granulocytes into the dermis and epidermis, associated with activation of NF-kappaB and increased expression of several proinflammatory cytokines and chemokines in skin of the dCKO mice. We further found that cultured FIP200 KO keratinocytes showed reduced NF-kappaB phosphorylation in response to tumor necrosis factor alpha stimulation, suggesting a paracrine regulation of aberrant NF-kappaB activation in the skin microenviroment of dCKO and FIP200 CKO mice. Together, these results demonstrate that ablation of FIP200, although not promoting tumorigenesis, can lead to skin inflammatory disorders, suggesting a novel function of FIP200 in the maintenance of normal skin homeostasis in vivo.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

A Nonautonomous Role for Retinal Frizzled-5 in Regulating Hyaloid Vitreous Vasculature Development

Frizzled-5 (Fzd5) is expressed in the developing retina of multiple species and appears to play species-specific roles during eye development. The present study analyzed the effects of tissue-specific deletion of Fzd5 on mammalian eye development. To generate Fzd5 conditional knockout (CKO) mice, Fzd5(+/-) mice carrying the Six3-Cre transgene were crossed with Fzd5(LoxP/LoxP) mice. To determine which cell lineages in the eye had Cre recombinase activity, Six3-Cre transgenic mice were crossed with ROSA-26 reporter mice, and lacZ activity was assayed. Histologic analysis, immunofluorescence, and TUNEL labeling were performed from embryonic day (E)12.5 to postnatal stages to analyze vascularization, cell proliferation, retinal organization, and apoptosis. On conditional disruption of Fzd5 specifically in the retina, but not in vitreous hyaloid vasculature (VHV), an abnormal accumulation consisting of pericytes and endothelial cells was observed in the vitreous as early as E12.5. The abundant retrolental cells persisted into postnatal stages and appeared as a pigmented intravitreal mass. In Fzd5 CKO mice there was failure of normal apoptosis of the VHV, and cells in the persistent VHV were maintained in the cell cycle up to postnatal day 23. Moreover, morphogenesis of the retina adjacent to the vasculature was disrupted, leading to retinal folds, detachment, and abnormal lamination. This phenotype is similar to that of human eye disease persistent hyperplastic primary vitreous (PHPV). Selective loss of Fzd5 in the retina results in PHPV and retinal defects through an apparently cell-nonautonomous effect, revealing a potential requirement for retina-derived signals in regulating the development of the VHV.

In vitro Transfer von Genen in spinale Gewebe

It is well known that cytokines are involved in the induction of intervertebral disc and articular cartilage destruction. Therapeutic proteins are of great potential as locally produced drugs after transfer of their cognate genes to the sites of interest. Chondrocytic cells from bovine os coccygis and discal chondrocytes from 6 wistar rats were isolated and cultured in vitro. The bacterial beta-galactosidase (LacZ) gene and the cDNA of the human interleukin-1 receptor antagonist (IL-1ra) were introduced into the cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1ra protein was determined by ELISA. Our study confirms that isolation and cultivation of bovine chondrocytic end plate cells and of rat discal cells in possible. Transfer of both LacZ and of the IL-1ra cDNA to cultured cells was successfull. The introduction of exogenous therapeutical genes into cells from the intervertebral disc and the end plate opens the possibility for a local gene therapy of IVD degeneration. This therapy has the potential to be specific, effective and appropriate to the chronicity of the disease.

The prpZ gene cluster encoding eukaryotic-type Ser/Thr protein kinases and phosphatases is repressed by oxidative stress and involved in Salmonella enterica serovar Typhi survival in human macrophages

The prpZ gene cluster consists of three ORFs coding for proteins with homology to eukaryotic-type Ser/Thr protein phosphatases 2C (prpZ) and Ser/Thr protein kinases (prkY and prkX). This cluster is present in the sequenced genomes of Salmonella enterica serovar Typhi (S. Typhi) strains Ty2 and CT18. This study investigated the genetic organization of this gene cluster, its regulation and its putative involvement in virulence. The three genes are transcribed as a polycistronic mRNA as demonstrated by reverse transcriptase (RT)-PCR. Analysis of a prpZ::lacZ transcriptional fusion showed that the prpZ locus is expressed throughout the growth phase. LacZ activity and real-time RT-PCR showed that transcription of the mRNA is negatively regulated upon exposure of cells to HOCl and, to a lesser extent, hydrogen peroxide. A deletion mutant of the prpZ gene cluster showed a significantly lower level of survival than the parental strain Ty2 in human macrophages at 48 h postinfection. Together these data suggest that prpZ, prkY and prkX are virulence genes that may be part of a signaling pathway controlling long-term survival of S. Typhi in host cells.

ArnR, a novel transcriptional regulator, represses expression of the narKGHJI operon in Corynebacterium glutamicum.

The narKGHJI operon that comprises putative nitrate/nitrite transporter (narK) and nitrate reductase (narGHJI) genes is required for the anaerobic growth of Corynebacterium glutamicum with nitrate as a terminal electron acceptor. In this study, we identified a gene, arnR, which encodes a transcriptional regulator that represses the expression of the narKGHJI operon in C. glutamicum cells under aerobic conditions. Disruption of arnR induced nitrate reductase activities of C. glutamicum cells and increased narKGHJI mRNA levels under aerobic growth conditions. DNA microarray analyses revealed that besides the narKGHJI operon, the hmp gene, which encodes flavohemoglobin, is negatively regulated by ArnR under aerobic conditions. Promoter-reporter assays indicated that arnR gene expression was positively autoregulated by its gene product, ArnR, under both aerobic and anaerobic conditions. Electrophoretic mobility shift assay experiments showed that purified hexahistidyl-tagged ArnR protein specifically binds to promoter regions of the narKGHJI operon and the hmp and arnR genes. A consensus sequence, TA(A/T)TTAA(A/T)TA, found in the promoter regions of these genes was demonstrated to be involved in the binding of ArnR. Effects on LacZ activity by deletion of the ArnR binding sites within the promoter regions fused to the reporter gene were consistent with the view that the expression of the narKGHJI operon is repressed by the ArnR protein under aerobic conditions, whereas the expression of the arnR gene is autoinduced by ArnR.

Whole Mount Enzyme Histochemistry as a Rapid Screen at Necropsy for Expression of β-Galactosidase (LacZ)–Bearing Transgenes: Considerations for Separating Specific LacZ Activity from Nonspecific (Endogenous) Galactosidase Activity

Whole mount enzyme histochemistry to localize lacZ-bearing transgenes (lacZ-WMH) also detects endogenous beta-galactosidases. The experiments reported here evaluated lacZ-WMH as a potential tool for transgene expression analysis during high-throughput rodent necropsies. A lacZ-WMH survey of organs from adult, wild-type, male and female mice (C57BL/6, FVB/N) and female rats (Sprague-Dawley) performed at the optimal pH (> or = 7.0) for bacterial lacZ yielded intense endogenous staining in the gonads, kidney, male accessory sex organs, salivary glands, submucosal glands in the duodenum, and thyroid. Substantial staining occurred in the adrenal cortex, lymph nodes, and linings of the gastrointestinal tract, the urinary bladder and uterus, and (for rat only) in the adenohypophysis, bone marrow, thymus, and trigeminal ganglia. Endogenous galactosidases were distributed similarly in sections of flash-frozen organs used for slide-based lacZ histochemistry (lacZ-SBH) at pH < or = 5.0 (optimal for eukaryotic enzymes). Cerebral neurons were labeled only by lacZ-SBH. At pH 7.4, endogenous but not specific lacZ activity was abolished for lacZ-SBH, while endogenous activity was not halted without reducing specific activity for lacZ-WMH. These data demonstrate that lacZ-WMH is feasible during rodent necropsies for many but not all organs if species-, strain-, and sex-specific divergence in endogenous galactosidase activity is considered and special fixation (3% paraformaldehyde for 3 hours at 4 degrees C) is used.

Expression pattern ofPopdc2 during mouse embryogenesis and in the adult

Whole-mount staining of beta galactosidase activity in a heterozygous E15.5 Popdc2-LacZ embryo. After staining the embryo was cleared in benzyl benzoate to visualize internal structures. Differentiated skeletal muscle tissue as well as the umbilical vessels display strong LacZ activity. Expression of the marker gene in the heart and in smooth muscle tissue of the gastrointestinal tract and the coelomic wall are not visible here. See Froese and Brand, Developmental Dynamics 237:762–769, 2008. © 2008 Wiley-Liss, Inc.

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.

Nuclear Factor 1 and T-Cell Factor/LEF Recognition Elements Regulate Pitx2 Transcription in Pituitary Development

Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Integral assessment of estrogenic potentials of sediment-associated samples

Exogenic endocrine-active substances are also called 'Endocrine Disrupting Chemicals' (EDC). They imitate or hinder the function of natural endogenic hormones or disturb the synthesis or the metabolism of hormones or of hormone receptors. The Enzyme-Linked Receptor Assay (ELRA) can detect estrogenic and anti-estrogenic effects at the level of receptor binding and is a useful tool for the integrative detection of contaminant effects. Although the test system has been used repeatedly in sediment assessments, the questions have remained concerning how it responds to variations in the physico-chemical matrix. For some bioassays, the salinity of the sample is a critical factor. This is especially relevant when testing wastewater samples or when sediment-associated samples in the tidal reaches of rivers are tested. Sediments in the tidal reaches of rivers change their salinity several times a day. Against this background, it would be beneficial to have a test procedure of known salinity tolerance. On account of this, the salinity tolerance of the ELRA was tested, assessed with reference substances at several salinity levels, and compared with the E-Screen method and a Yeast Estrogen Screen (YES), which are also frequently applied in environmental testing. The aim of this paper was to explore when the salinity limits within these test procedures are applicable. The trials should reveal the working range to be expected, characterize the salinity-dependent variations in sensitivity of the test, and provide options for methodological adjustments to improve the stability against increased salinity. The ELRA was carried out with the human Estrogen Receptor alpha. (ER) using the same principle like a competitive immunoassay based on ligand-protein interaction. However, an essential difference is the use of a physiologically relevant receptor instead of an antibody as a linking protein. The ELRA measures the competition of sample estrogens and anti-estrogens against estradiol supplied as a BSA-coating conjugate for the binding site of dissolved ER. Estradiol or xeno-estrogen binding is quantified by a biotynilated anti-ER antibody and the subsequent measurement of peroxidase activity by a streptavidin-POD-biotin complex. The E-Screen was performed with the human breast cancer cell line MCF-7, which expresses the estrogen receptor constitutively. Cell proliferation depends on binding of estrogens or xeno-estrogens with the receptor. After incubation, estrogen-dependent cell growth was measured by sulforhodamin B staining. The YES was performed with a recombinant yeast strain, transfected with a receptor and a reporter plasmid bearing the estrogen receptor and a vitellogenin gene fused with the reporter gene lacZ. Estrogen or xeno-estrogen-dependent gene induction was measured indirectly by LacZ activity. The salinity levels were simulated in varying concentrations with NaCl from 0 to 40 per thousand or Artificial Sea Water (ASW) from 0 to 32 per thousand. The study characterized the factor 'salinity' for the prospective application fields of the ELRA. With reference substances such as 17-beta-estradiol, the ELRA showed classical sigmoidal concentration-effect relations in a range from 0.05 to 100 microg/l under physiological conditions. After a methodological adjustment to compensate decreasing receptor-binding affinity of estrogens and xeno-estrogens at higher salinity levels, the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. In tests, the ELRA reached under the influence of salinity a mean limit of detection of 0.062 microg/l 17-beta-estradiol. The mean relative inter-test error was around 11%. Above concentrations of 20.5 per thousand there is a risk of false negative assessment. Compared with the E-Screen method using the MCF7 cell line and the yeast estrogen test system (YES), the ELRA shows a lower sensitivity to 17-beta-estradiol. In the E-Screen, the cell proliferation was strongly reduced by sodium chloride induced cytotoxicity. In comparison with the E-Screen, the salinity tolerance of the YES and YAS methods is significantly higher. Despite adaption, total salinity tolerance could not be achieved with the ELRA. Freshwater samples were generally appraisable. Higher salinity levels above 20.5 per thousand would tend towards false negative results. The low inter-test error of 11% makes the ELRA suitable for the detection of estrogenic and anti-estrogenic potentials of single substances, substance mixtures, and of environmental samples. The ELRA is very fast and reproducible, it can be used for high-throughput screening in a microplate format at low cost, it is robust to microbial contamination, and is less susceptible to cytotoxic interferences than cell culture methods. In their established form, the YES and the E-Screen methods are not applicable for liquid phase testing at higher salinity conditions. The salinity-adapted test version of the ELRA described here shows a broader working range for samples. Native water samples of more or less brackish origin or high-salinity effluent samples are testable. Results of tests with sediment associated samples of different salinity will be subject of a forthcoming publication.

Transcriptional organization of thePorphyromonas gingivalis fimAlocus

Two different transcriptional start sites, as well as promoter regions and translational starts, have been proposed for the fimA gene encoding a long fimbriae subunit protein of Porphyromonas gingivalis. In this study, the fimA promoter regions and organization of the fimA operon were characterized. The two putative promoter regions for fimA were fused with a lacZ reporter gene, cloned into the shuttle plasmid vector pT-COW, and the recombinant plasmids were introduced to P. gingivalis 33277. Reverse transcriptase-polymerase chain reaction demonstrated mRNA production from the promoter proximal to the translational start. LacZ activities of P. gingivalis containing the recombinant plasmids showed that maximal expression of fimA was promoted by the proximal promoter in combination with distal regulatory sequences. A polycistronic message spanning PG2130, PG2131 and fimA (PG2132) was observed, thus fimA transcripts may also be generated by processing of the polycistronic message.