Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.

AIMS: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. METHODS AND RESULTS: For this purpose, we established a protocol for the transformation of the plasmid pAD43-25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re-germination. CONCLUSIONS: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP-tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re-germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.

Country-specific effects of neonicotinoid pesticides on honey bees and wild bees.

Neonicotinoid seed dressings have caused concern world-wide. We use large field experiments to assess the effects of neonicotinoid-treated crops on three bee species across three countries (Hungary, Germany, and the United Kingdom). Winter-sown oilseed rape was grown commercially with either seed coatings containing neonicotinoids (clothianidin or thiamethoxam) or no seed treatment (control). For honey bees, we found both negative (Hungary and United Kingdom) and positive (Germany) effects during crop flowering. In Hungary, negative effects on honey bees (associated with clothianidin) persisted over winter and resulted in smaller colonies in the following spring (24% declines). In wild bees (Bombus terrestris and Osmia bicornis), reproduction was negatively correlated with neonicotinoid residues. These findings point to neonicotinoids causing a reduced capacity of bee species to establish new populations in the year following exposure.

Differential effects of DNA-binding proteins on bidirectional transcription from the common promoter region of human collagen type IV genes COL4A1 and COL4A2.

Expression of the heterotrimeric collagen IV (alpha 1(IV))2 alpha 2(IV) is essential for the structural integrity and functional properties of basement membranes. The genes COL4A1 and COL4A2 coding for both subunits are located close to each other on the same chromosome and are transcribed from a common bidirectional promoter element. Binding of at least three different nuclear proteins could be detected within this promoter, a CCAAT-binding protein, Sp1 and a newly identified factor, designated 'CTCBF'. Mutagenesis of binding sites proved that these factors are essential for the efficient transcription of both genes, but revealed differential gene-specific effects. Therefore, the common promoter region of collagen IV does not represent an equally functional bidirectional element, but may be better understood as two overlapping gene-specific promoters with shared elements.

Signaling by epidermal growth factor differentially affects integrin-mediated adhesion of tumor cells to extracellular matrix proteins.

The adhesion of different epidermal growth factor (EGF) receptor (EGFR) expressing cell lines to various extracellular matrix (ECM) proteins is influenced by EGF. To investigate a putative receptor crosstalk between EGFR and integrins we chose two cell lines for a more detailed analysis: the highly metastatic rat mammary carcinoma clone MTLn3 that showed increased adhesion to a panel of ECM proteins in the presence of 10 ng/ml EGF and the nonmetastatic human vulva carcinoma cell line A431 which showed a decreased adhesion under the same conditions. These EGF-mediated stimulatory or inhibitory effects on adhesion were observed within a few minutes. On human A431 cells the inhibitory effect was blocked by an EGFR specific antibody that interferes with ligand binding. In cell adhesion assays performed in the presence of divalent cations MTLn3 and A431 cells exhibited the typical behavior described for integrin-dependent matrix adhesion: Mn2+ enhanced binding to collagen IV and fibronectin whereas Ca2+ inhibited adhesion to collagen IV but not to fibronectin. Adhesion-inhibition assays with anti-human integrin antibodies revealed that A431 cells adhere to collagen via alpha 1 beta 1 and alpha 2 beta 1, and that adhesion to fibronectin is mediated predominantly through alpha 5 beta 1. The interaction of MTLn3 cells with fibronectin was in part RGD dependent, indicating the involvement of either alpha 3 beta 1 or alpha 5 beta 1. Addition of EGF in these assays showed that affecting the integrin extracellular domains by addition of either bivalent cations, RGD peptides, or function-blocking integrin antibodies did not prevent the effects mediated by EGF. We conclude that signals downstream of EGFR can modulate integrin-mediated adhesion to ECM proteins in both an inhibitory and a stimulatory manner.

Effect of bosentan on NF-kappaB, inflammation, and tissue factor in angiotensin II-induced end-organ damage.

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells.

To establish a nonradioactive method for demonstrating HPV DNA in routinely treated smears of the uterine cervix (alcohol fixation, staining according to Papanicolaou, preservation), in situ hybridizations were carried out in HeLa and SiHa cells grown on slides. After detailed investigations, the sensitivity and specificity of the biotin-avidin method (10) initially used proved to be inadequate for this purpose. Demonstration of HPV 16 DNA in SiHa cells (SiHa cells only contain 1-2 HPV genome copies) was possible only by use of digoxigenin-labeled HPV 16 gene probes, as well as an improved purification of the sample DNA from vector contaminations. Thus, for the first time a protocol for correlation of the results of an in situ hybridization with the cytological appraisal in the very same smear preparation has been developed for routine diagnostics.

Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus.

Human tonsillar and adenoid tissues from surgical specimens and cell cultures established therefrom were screened for adenovirus type 2 (Ad2) sequences by in situ hybridization. For labeling we have utilized biotinylated DNA probes. We report detection of adenoviral sequences after hybridization with adenovirus type 2 DNA probes in tissues as well as in cell cultures from specimens without any signs of infectious virus even after long-term cultivation. In the infected tonsils only some of the cells appear to carry viral sequences. In conclusion, truly latent adenovirus infections in man seem to occur.

American foulbrood of the honey bee: occurrence and distribution of different genotypes of Paenibacillus larvae in the administrative district of Arnsberg (North Rhine-Westphalia).

Between March 2003 and October 2004, Paenibacillus larvae, the aetiological agent of American foulbrood disease of the honey bee, was isolated from broodcombs and honey samples of 54 apiaries in the administrative district of Arnsberg (North Rhine-Westphalia, Germany). Genotyping of 176 P. larvae isolates with repetitive element polymerase chain reaction fingerprinting (rep-PCR) using BOX A1R and MBO REP1 primers revealed five different genotypes (AB, Ab, ab, ass, Acapital BE, Cyrillic). In samples of three apiaries, more than one genotype was detected. A combination of two genotypes was isolated from honey samples of the same hive two times (ab/ass and Ab/ab). The five genotypes were not randomly distributed in the district, but revealed a certain geographical clustering. Possible factors with impact on the genotype diversity and the distribution pattern are discussed.

Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways.

Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-(alpha) (TNF(alpha)) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNF(alpha) caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNF(alpha) stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.

Purification of the sequence-specific transcription factor CTCBF, involved in the control of human collagen IV genes: subunits with homology to Ku antigen.

The common promoter region of the human collagen type IV genes COL4A1 and COL4A2 comprises a C5TC7 sequence ('CTC box') which is specifically recognized by the recently identified transcription factor CTC box binding factor (CTCBF) involved in the control of divergent transcription of the two genes. This factor has now been purified by affinity chromatography on heparin-agarose and CTC-Sepharose. The CTCBF contains two subunits, CTC75 and CTC85, with molecular weights of 75 and 85 kDa, respectively. Sequence analysis of LysC-derived peptides of the two subunits revealed identity or close homology to p70 and p80 subunits of the human autoantigen Ku. The sequence-specific binding CTCBF represents a presumably tetrameric complex composed of two CTC75/85 heterodimers with an apparent molecular weight of 360-400 kDa. UV crosslinking experiments, the use of Ku-specific antibodies in gel retardation assays and immunoblotting proved that both subunits are involved in sequence-specific interaction with the CTC box motif. The tetrameric complex dissociates in a concentration-dependent manner to CTC75/85 heterodimers which now bind sequence independently to DNA. Three lines of evidence indicate that TATA binding protein (TBP) is additionally involved in the formation of CTCBF: (i) TBP can be detected in purified CTCBF; (ii) the addition of recombinant TBP stimulates formation of the CTCBF-DNA complex; and (iii) antibodies directed against TBP interfere strongly with the formation of the specific protein-DNA complex. The results presented support the idea that the subunits CTC75 and CTC85 (identical or homologous to p70 and p80 of the Ku antigen) are integral parts of CTCBF, and give a first indication of the importance of TBP in the formation of CTCBF.

Prevention of EGF-modulated adhesion of tumor cells to matrix proteins by specific EGF receptor inhibition.

The adhesion of tumor cells to various extracellular matrix (ECM) proteins is influenced by epidermal growth factor (EGF). Maximal effects are obtained at low EGF concentrations, at which mostly the cytoskeleton-associated high-affinity EGF receptors (EGFRs) are saturated. Tumor cells expressing EGFR either endogenously (MDA MB 231, MTLn3) or, for the human EGFR, ectopically (MTC HER1/1) in intermediate amounts exhibited, upon EGF addition, increased cellular adhesion to various ECM proteins, such as fibronectin, collagens and vitronectin. In contrast, human A431 and MDA MB 468 cells, over-expressing EGFR, demonstrated reduced attachment in similar experimental conditions. Both increased as well as reduced EGF-dependent adhesion could be blocked using either ligand-blocking monoclonal antibody 14E1 or the potent EGFR tyrosine kinase inhibitor PD 153035. Our data indicate that signals downstream of EGFR activation are responsible for the opposing effects of EGF on cellular adhesion since both can be prevented by EGFR inhibition. Thus, the integration of EGFR- and integrin-dependent signals can be different in carcinoma cell lines and might be influenced by EGFR numbers.