Automatic control of the effluent turbidity from a chemically enhanced primary treatment with microsieving.

For chemically enhanced primary treatment (CEPT) with microsieving, a feedback proportional integral controller combined with a feedforward compensator was used in large pilot scale to control effluent water turbidity to desired set points. The effluent water turbidity from the microsieve was maintained at various set points in the range 12-80 NTU basically independent for a number of studied variations in influent flow rate and influent wastewater compositions. Effluent turbidity was highly correlated with effluent chemical oxygen demand (COD). Thus, for CEPT based on microsieving, controlling the removal of COD was possible. Thereby incoming carbon can be optimally distributed between biological nitrogen removal and anaerobic digestion for biogas production. The presented method is based on common automation and control strategies; therefore fine tuning and optimization for specific requirements are simplified compared to model-based dosing control.

Activation of NF-kappaB in the mouse spinal cord following sciatic nerve transection.

NF-kappaB is a ubiquitous nuclear transcription factor that regulates a number of physiological processes. NF-kappaB activity has been implicated in enhancing neuronal survival following CNS injury. The present study was conducted to test the hypothesis that NF-kappaB activity is up-regulated in neurons of the spinal cord in response to peripheral nerve transection. In this series of experiments, we used NF-kappaB reporter mice in which activation of NF-kappaB drives the expression of the lac-z gene. The response to injury of cells in the spinal cord was assessed by evaluating the number and distribution of beta-galalactosidase (beta-gal)-positive cells following sciatic nerve transection. The animals were randomly assigned to four groups, which were allowed to survive for one, three, five and ten days. Four mice that did not undergo sciatic nerve transection were assigned to each group to serve as controls. The total number of beta-gal-positive cells in the right and left dorsal and ventral horns were compared. The numbers of beta-gal-positive cells between the right and left sides were significantly different three and five days post axotomy (p<0.05). Double immunofluorescent labeling was utilized to characterize which cells showed NF-kappaB activity, and it revealed that all beta-gal-positive cells were colocalized with MAP-2-positive neurons. The results of this study demonstrated that complete sciatic nerve transection leads to an up-regulation of NF-kappaB transactivation in spinal neurons ipsilateral to the side of transection. The increase in activity in the ipsilateral dorsal horn is consistent with this transcription factor acting as neuronal survival signal during this time frame in response to the peripheral nerve insult.

Injury-induced NF-kappaB activation in the hippocampus: implications for neuronal survival.

Nuclear factor (NF)-kappaB p50 protein is involved in promoting survival in hippocampal neurons after trimethyltin (TMT)-injury. In the current study, hippocampal NF-kappaB activity was examined and quantitated from transgenic kappaB-lacZ reporter mice after chemical-induced injury. NF-kappaB activity was localized primarily to hippocampal neurons and significantly elevated over that in saline-treated mice between 4 and 21 days after TMT injection. Seven days after TMT injection, a timepoint of elevated NF-kappaB activity, gene expression in the hippocampus was studied by microarray analysis through comparison of expression profiles between treated nontransgenic and p50-null mice with their saline-injected controls. Seventeen genes increased in nontransgenic TMT-treated mice relative to saline-treated as well as showing no increase in p50-null mice, indicating a role for p50 in their regulation. One of these genes, the Na+, K+-ATPase-gamma subunit, was detected in brain for the first time. Several of the genes modulated by NF-kappaB are potentially related to neuroplasticity, providing additional evidence that this transcription factor is a neuroprotective signal in the hippocampus.

NF-kappa B is developmentally regulated during spermatogenesis in mice.

To analyze NF-kappa B activity in the testis, we used murine transgenic lines carrying a LacZ reporter gene under the control of a NF-kappa B-responsive promoter (Schmidt-Ullrich et al. [1996] Dev 122:2117-2128). We constructed three independent lines containing the promoter of the gene encoding p105, the precursor of the p50 subunit. This promoter contains three NF-kappa B-binding sites in its proximal part. Our results show that in adult mice, the beta-galactosidase activity which reflects nuclear NF-kappa B activity, is first detected in spermatocytes at the pachytene stage and remains activated in the following steps of germ cell differentiation and maturation. Using transgenic mice carrying a p105nlslacZ construct with the 3 NF-kappa B sites mutated in the p105 promoter, we found a significant reduction in the transgene activity, confirming the important role of NF-kappa B in the activation of the transgene. To confirm the stage of induction during spermatogenesis, we analysed the beta-galactosidase activity in the testes from prepuberal mice in which cells synchrouneously enter meiosis. We detected the transgene activity at 18 days after birth, corresponding to the pachytene stage in spermatocytes. In nuclear extracts prepared from prepuberal mice, we found a peak of NF-kappa B DNA-binding activity made of p50 and p65 subunits at day 18 after birth, which remains high in the later stages. Further analysis showed that I kappa B alpha and beta, but not epsilon are expressed in the testes. Altogether, these data suggest that NF-kappa B factors are stage specifically controlled and may play a role during the development of sperm cells.

Complete lack of NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation.

NF-kappaB activity is induced by cytokines, stress, and pathogens. IKK1 and IKK2 are critical IkappaB kinases in NF-kappaB activation. In this study mice lacking IKK1 and IKK2 died at E12. Additional defect in neurulation associated with enhanced apoptosis in the neuroepithelium was also observed. MEF cells from IKK1(-/-)/IKK2(-/-) embryos did not respond to NF-kappaB inducers. Upon crossing with kappaB-lacZ transgenic mice, double-deficient embryos also lost lacZ transgene expression in vascular endothelial cells during development. Our data suggest that IKK1 and IKK2 are essential for NF-kappaB activation in vivo and have an important role in protecting neurons against excessive apoptosis during development.

In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-kappaB/Rel complexes.

To analyze the NF-kappaB/Rel activity pattern in a living organism, we previously generated transgenic mice carrying a kappaB-dependent lacZ gene. In situ analysis of both primary and secondary lymphoid organs revealed a strong NF-kappaB transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula with very little activity in lymphocytes and thymocytes. Using fluorescein-di-beta-D-galactopyranoside (FDG) as a vital substrate for the beta-galactosidase, we re-examined by flow cytometry the NF-kappaB/Rel transcriptional activity in our mouse model. We report here that such constitutive NF-kappaB/Rel activity was significantly detected in thymocytes at the CD44+CD25(-) stage. This constitutive activity extended with CD25 expression to the majority of the CD44(-)CD25(+) thymocytes and was then restricted to a few mature T cells. In the spleen, constitutive NF-kappaB/Rel activity was found in most B cells, unlike T cells which were largely negative. Virgin IgD(+) B cells expressed higher levels of NF-kappaB transcriptional activity than other B cell types. Altogether, these results suggest that NF-kappaB/Rel complexes are key players in the in vivo differentiation of IgD(+) B lymphocytes and possibly CD25(+) thymocytes.

IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis.

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.

Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines.

The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene. NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant. Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.

NF-kappaB activity in transgenic mice: developmental regulation and tissue specificity.

The transcription factor family NF-kappaB/Rel is responsible for the regulation of a large number of cellular genes and some viruses. Since there is a strong similarity between the NF-kappaB/Rel family members and the Drosophila melanogaster protein DORSAL, which is activated early during embryogenesis, we were interested in determining the pattern of NF-kappaB activity during mouse development. Two lacZ reporter constructs, each driven by promoter elements that are dependent on the presence of nuclear NF-kappaB/Rel activity, were used to produce transgenic mice. The analysis of these mice did not identify nuclear NF-kappaB/Rel activity in early development prior to implantation or during the gastrulation processes. Earliest expression of the lacZ transgene was detected on day E12.5. Before birth lacZ expression was seen in discrete regions of the rhombencephalon of the developing brain, in the spinal medulla, in some of the blood vessels and in the thymus. After birth, the NF-kappaB/Rel activity in the thymus remained but nuclear activity was also found in the bone marrow, in the spleen and in the capsule of the lymph nodes. In the central nervous system, drastic changes in NF-kappaB/Rel activity could be observed in the first 3 weeks after birth, when the cortex and the cerebellum reach functional and morphological maturity. Considering the results of the p50, p65, relB and c-rel knock-out mice and our present findings, we believe that the NF-kappaB/Rel proteins known so far are probably not implicated in processes of early development and differentiation of the different tissues, but rather in maintaining their function once matured.

Differential kinetics of polypeptide expression and different biological activities in the human fibroblast response to dsRNA or interferon treatment.

Using two-dimensional electrophoresis on total and nuclear extracts of human fibroblasts, we compared polypeptide patterns of cells treated with interferon-beta (IFN-beta), IFN-gamma, or with dsRNA in the presence of anti-IFN antibodies. The analysis of whole-cell extracts revealed that, after a 6-h treatment, the three agents induce the synthesis of a common set of proteins in addition to others that are specifically induced either by IFNs or by dsRNA. After a 15-h treatment, this common set of proteins was only induced by IFNs. Furthermore, at this time, IFNs also regulated proteins whose synthesis was specifically induced or repressed by poly(I).poly(C) in the 6-h treated cells. These results indicate that poly(I).poly(C) regulates protein expression more rapidly and more transiently than IFNs. The analysis of nuclear extracts showed similar differential kinetics of protein expression. However, a greater number of polypeptides was found to have their synthesis specifically induced by dsRNA. Moreover, poly(I).poly(C) was found to be mitogenic in these cells and did not induce a significant resistance to vesicular stomatitis virus (VSV). This study provides evidence for an overlap in the expression of proteins by dsRNA and IFNs, although these compounds do not share the same biological activities.

Post-transcriptional regulation of transferrin receptor mRNA by IFN gamma.

IFN gamma inhibits the rise in transferrin receptor mRNA level which is normally observed when stationary WISH cells are stimulated to proliferate. This effect is not attributable to a change in the transcription rate of the transferrin receptor gene or in the cytoplasmic stability of the mRNA. The IFN gamma-induced reduction of the transferrin receptor mRNA content is already present at the nuclear level to an extent comparable to that observed in whole cells. Thus, IFN gamma does not impair the passage of this mRNA from the nuclear to the cytoplasmic compartment but probably interferes with a nuclear post-transcriptional event during the processing of the immature transferrin receptor mRNA. Two different levels of regulation of transferrin receptor mRNA have been previously reported. Iron modulates the cytoplasmic stability of this mRNA through the binding of a specific cytoplasmic factor, whereas cell growth variation influences the transcription of this gene. Our results suggest the existence of another mechanism of regulation for transferrin receptor gene expression not so far considered. Furthermore, the distinction between the mechanism of regulation exerted by IFN gamma and that exerted by cell proliferation on transferrin receptor gene expression suggests that, in WISH cells, the IFN-induced transferrin receptor decay is not a consequence of cell growth arrest but rather one of the causes of the antiproliferative effect of IFN through iron deprivation.

Direct induction of interferon-gamma- and interferon-alpha/beta-inducible genes by double-stranded RNA.

Using Northern analysis, we here show that the inducibility by double-stranded (ds) RNA of interferon-alpha/beta-inducible genes is not restricted to a few genes but extends to all the genes known to be stimulated by IFN type I in fibroblasts. Moreover, we show that some genes, preferentially regulated by IFN-gamma, are also activated by dsRNA. We present a series of arguments demonstrating that the induction by dsRNA is not mediated by IFN. In addition to the fact that this induction occurs in the presence of cycloheximide and/or anti-IFN-alpha/beta antibodies in fibroblasts, we observed that, in IFN-resistant Daudi cells, ISG15 and IP-10 genes which are not induced by IFN-beta, are still inducible by dsRNA. dsRNA is also still active on 2-5 AS and ISG15 genes in cells carrying homozygous deletions of IFN alpha/beta genes. Actinomycin D experiments and nuclear in vitro elongation assays reveal that the induction by dsRNA involves, as its early step, a transcriptional event. This induction was found not to require protein synthesis, suggesting that activation of preexisting cellular factors is involved. The opposite inducibility by dsRNA of IFN-beta and 2',5'-oligoadenylate (2-5A) synthetase genes in serum-deprived fibroblasts indicates that pathways of induction by dsRNA of these two genes are not identical. Inhibition by 2-aminopurine of the induction of IFN-inducible mRNAs by IFN-beta or dsRNA suggests the participation of a protein kinase in their mechanism of action.

Tumour necrosis factor enhances induction by beta-interferon of a ubiquitin cross-reactive protein.

Tumour necrosis factor alpha (TNF-alpha) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-alpha also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-alpha induction of UCRP occurred at the level of transcription. TNF-alpha induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-beta-interferon (IFN-beta) antibody. However no measurable increase in the mRNA specific for IFN-beta was detected after TNF-alpha treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-beta antibody indicates that these cells are making IFN-beta already. We conclude that the TNF-alpha induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-beta in the responding cells. Furthermore TNF functions to enhance the existing IFN-beta activity.

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).

Trypanosoma brucei contains two RNA polymerase II largest subunit genes with an altered C-terminal domain.

We have identified and cloned four trypanosomal RNA polymerase largest subunit genes. Here, we present the molecular analysis of two genes, Trp4.8 and Trp5.9. The sequence of these genes shows that they are almost identical to each other and indicates that they encode the largest subunit of RNA polymerase II. Both genes contain a C-terminal extension that is clearly distinct from that of other eukaryotic RNA polymerase II genes, because it lacks the common tandemly repeated heptapeptide sequence and is rich in acidic amino acids. It shares many potential phosphorylation sites, however, with the C-terminal extension of other eukaryotic RNA polymerase II large subunits. The presence of two RNA polymerase II loci suggests that a fourth RNA polymerase could be formed. Interestingly, the fourth gene is only found in species exhibiting antigenic variation.

RNA polymerases B and C are more closely related to each other than to RNA polymerase A.

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.

Conditional mutants of RPC160, the gene encoding the largest subunit of RNA polymerase C in Saccharomyces cerevisiae.

A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO3 and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pcil-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation.

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A.

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.

Isolation of structural genes for yeast RNA polymerases by immunological screening.

A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.