Biomedical subjects

H Hauser

Publications and source records attributed to H Hauser.

At least 19 recordsLinked to original sources

PubMed2006

A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.

Animals↗

PubMed2006

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

Cell Line↗

PubMed2005

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

ATP-Binding Cassette Transporters↗

PubMed2004

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Base Sequence↗

PubMed2004

Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines.

DNA vaccines are an appealing strategy for inducing cytotoxic T-lymphocyte and antibody responses against tumor cells as well as infectious agents. Dendritic cells (DCs) play a critical role in inducing immune responses, but their potential is not fully utilized in the DNA vaccine setting since they take up only a minor fraction of the injected DNA. Here we describe a novel DNA vaccination strategy based on the targeting of a modified tumor-associated antigen, the human papilloma virus (HPV) type 16 E7 protein, to DCs by a heat-shock protein (HSP) to enhance antigen presentation and immune responses. Specifically, a chimerical HPV-E7 and HSP70 fusion gene preceded with a leader sequence was constructed. When mice were immunized with this construct, the DNA is taken up by various types of cells, which then produce and secrete an HPV-E7-HSP70 fusion protein that is targeted to DCs by the HSP70 portion of the chimerical molecule for antigen presentation. In studies to test the efficacy of this strategy, we demonstrated that DNA vaccination with this secretory HPV-E7-HSP70 construct strongly enhanced an antigen-specific CD8+ T-cell response as well as a specific B-cell response in mice. Furthermore, this immunization approach not only protected mice against lethal challenge with an HPV E7-expressing tumor line (TC-1), but also showed a therapeutic effect against established tumors. The results of this study indicate that secretory HSPs can be broadly used to target tumor-associated antigens to DCs to enhance antigen-specific immune responses.

Animals↗

PubMed2002

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13.

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.

Animals↗

PubMed2002

Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein.

OBJECTIVE: One of the main open questions in chondrocyte transplantation is the fate of the implanted cells in vivo. We intended to establish prerequisites for such studies in animal models and to show the feasibility of this approach in rabbits. Isolated articular chondrocytes were retrovirally marked using green fluorescence protein (GFP) as a cell-specific marker in order to allow an in vivo follow-up of these cells. METHODS: Chondrocytes from rabbits, sheep, cattle and humans were isolated and infected with murine leukemia virus-derived retroviruses carrying the GFP gene. The influence of the host range of three packaging cell lines (PA317, PT67, PG13), start cell concentrations, number of cell passages and number of infection cycles on the efficiency of infection was investigated. Stability of GFP expression was followed by FACS analysis, confocal imaging and fluorescence microscopy. For in vivo follow-up of GFP expression we used marked allogeneic chondrocyte populations grown on scaffold material and implanted them into full-thickness defects in knee joints of rabbits. RESULTS: Retroviruses from all three packaging cell lines were able to infect rabbit and human chondrocytes, whereas only retroviruses released from PG13 cells were able to infect sheep and bovine chondrocytes efficiently. Optimization of the infection with these viruses resulted in efficiencies of 60-90% GFP-expressing chondrocytes. Populations of 100% marked chondrocytes were obtained by cell sorting. GFP expression stability of such marked chondrocyte populations was followed in monolayer culture and in 3-D culture on different scaffold materials. The expression of GFP was stable on all tested materials for at least 4 weeks. In monolayer culture GFP expression was stable for more than 8 months. In vivo, we observed stable GFP expression in the transplants during a four-week time course. CONCLUSION: Retroviral GFP gene transfer led to long-term expression in chondrocytes from rabbits, sheep, cattle and humans. Transgene expression and the number of implanted chondrocytes remain stable for at least 4 weeks in vivo. This method permits a rapid monitoring of chondrocytes and provides a basis for following the fate of these cells in vivo.

Animals↗

PubMed2002

[Recovery coefficients for the quantification of the arterial input functions from dynamic PET measurements: experimental and theoretical determination].

AIM: For kinetic modelling of dynamic PET data, the arterial input function can be determined directly from the PET scans if a large artery is visualized on the images. It was the purpose of this study to experimentally and theoretically determine recovery coefficients for cylinders as a function of the diameter and level of background activity. METHODS: The measurements were performed using a phantom with seven cylinder inserts (Ø = 5-46 mm). The cylinders were filled with an aqueous 68Ga solution while the main chamber was filled with a 18F solution in order to obtain a varying concentration ratio between the cylinders and the background due to the different isotope half lives. After iterative image reconstruction, the activity concentrations were measured in the center of the cylinders and the recovery coefficients were calculated as a function of the diameter and the background activity. Based on the imaging properties of the PET system, we also developed a model for the quantitative assessment of recovery coefficients. RESULTS: The functional dependence of the measured recovery data from the cylinder diameter and the concentration ratio is well described by our model. For dynamic PET measurements, the recovery correction must take into account the decreasing concentration ratio between the blood vessel and the surrounding tissue. Under the realized measurement and data analysis conditions, a recovery correction is required for vessels with a diameter of up to 25 mm. CONCLUSIONS: Based on the experimentally verified model, the activity concentration in large arteries can be calculated from the measured activity concentration in the blood vessel and the background activity. The presented approach offers the possibility to determine the arterial input function for pharmacokinetic PET studies non-invasively from large arteries (especially the aorta).

Humans↗

PubMed2001

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

3T3 Cells↗

PubMed2001

Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine.

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.

Adenocarcinoma↗

PubMed2001

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

Animals↗

PubMed2001

Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement.

Successful retroviral vector construction is still empirical. Test systems for vector efficiency are based on statistical comparison of numerous infectants with single proviral integrates, since their expression depends on the chromosomal surroundings. More reliable data would be obtained if different vector constructs were studied in an identical chromosomal context. Here, we demonstrate the use of a new method, in which chromosomal sites are provirally tagged in such a way that they can be targeted with other expression cassettes. The original tagging integrate is replaced in one step by the targeting element. This permits a reliable comparison of different retroviral vector configurations, eliminating the influence of neighboring chromosomal elements. We compared different retroviral vector types for coexpression of two genes: a vector containing an internal promoter and a vector with an internal ribosome entry site (IRES) element. In contrast to bicistronic retroviral vectors, dual-promoter proviruses exhibited rapid inactivation of the long terminal repeat (LTR)-driven gene expression. Targeted exchange of the dual-promoter provirus with a bicistronic retroviral cassette resulted in gain of expression stability. The reverse experiment confirmed this promoter interaction phenomenon since initial expression stability from a single-promoter bicistronic provirus was lost by targeted exchange with a dual-promoter cassette. In addition, targeting exchange of the dual-promoter provirus, replacing the LTR with an artificial (Tet) promoter restored expression stability. These observations, valid for various integration sites, prove the strong interaction between the LTR and the internal promoter. Our results have implications for retroviral vector design and suggest that retroviral coexpression of two genes is more predictable in the bicistronic configuration.

3T3 Cells↗

PubMed2001

Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis and few therapeutic options. The aim of the study was to evaluate the potential of IFN regulatory factor-1 (IRF-1) for cytokine gene therapy of HCC using an IRF-1/human estrogen receptor fusion protein (IRF-1hER), which is reversibly activatable by beta-estradiol (E2). IRF-1hER stably expressing murine Hepa1-6 HCC cells (HepaIRF-1hER) were characterized by lowMHC 1, highCD54, and lack of MHC II, CD80, and CD86 expression. Activation of HepaIRF-1hER cells induced a highMHC I, lowMHC II, and highCD54 phenotype. Furthermore, they were characterized by IFN-beta secretion, decreased anchorage-independent growth in a soft agar assay, and diminished cell growth. Tumor growth in E2-treated syngeneic C57L/J mice, but not in E2-untreated mice, was suppressed. These E2-treated mice were protected against rechallenge with HepaIRF-1hER and wild-type Hepa1-6 tumors even in the absence of E2, suggesting induction of tumor specific immunity. In fact, significant CTL activity against Hepa1-6 tumors and the endogenously expressed HCC-specific self antigen alpha-fetoprotein was observed. Antitumoral effects, however, were only partially dependent on both CD4+ and CD8+ T cells. IRF-1 treatment of mice bearing HepaIRF-1hER tumors resulted in growth arrest of tumors, and a significant survival benefit was observed in comparison to E2-untreated mice. In conclusion, our data demonstrate that IRF-1 suppresses HCC growth through both a direct antitumor growth effect and enhanced immune cell recognition of the tumor and is a promising candidate for gene therapy of HCC.

Animals↗

PubMed2001

Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells.

Animals↗

PubMed2001

Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET.

We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab')(2) fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab') fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10(7) M(-1)) while the binding capacity of cells was high (8.4 x 10(6) BS-MAbs per cell). Tumor uptake of the 67Ga labeled chelate in pretargeted animals was to 5.8 +/- 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68Ga and 67Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68Ga chelate, clearly visualized all tumors.

Animals↗

PubMed2001

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1).

Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: "cen-WEE1-D11S2043-ZNF143-RANBP7-CEGF1- ST5-D11S932-LMO1-D11S572-TUB-tel", with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.

AT Rich Sequence↗

PubMed2000

Short-chain phospholipids as detergents.

The physico-chemical properties of short-chain phosphatidylcholine are reviewed to the extent that its biological activity as a mild detergent can be rationalized. Long-chain diacylphosphatidylcholines are typical membrane phospholipids that form preferentially smectic lamellar phases (bilayers) when dispersed in water. In contrast, the preferred phase of the short-chain analogues dispersed in excess water is the micellar phase. The preferred conformation and the dynamics of short-chain phosphatidylcholines in the monomeric and micellar state present in H(2)O are discussed. The motionally averaged conformation of short-chain phosphatidylcholines is then compared to the single-crystal structures of membrane lipids. The main conclusion emerging is that in terms of preferred conformation and motional averaging short-chain phosphatidylcholines closely resemble their long-chain analogues. The dispersing power of short-chain phospholipids is emphasized in the second part of the review. Evidence is presented to show that this class of compounds is superior to most other detergents used in the solubilization of membrane proteins and the reconstitution of the solubilized proteins to artificial membrane systems (proteoliposomes). The prominent feature of the solubilization/reconstitution of integral membrane proteins by short-chain PC is the retention of the native protein structure and hence the protein function. Due to their special detergent-like properties, short-chain PC lend themselves very well not only to membrane solubilization but also to the purification of integral membrane proteins. The retention of the native protein structure in the solubilized state, i.e. in mixed micelles consisting of the integral membrane protein, intrinsic membrane lipids and short-chain PC, is rationalized. It is hypothesized that short-chain PC interacts primarily with the lipid bilayer of a membrane and very little if at all with the membrane proteins. In this way, the membrane protein remains associated with its preferred intrinsic membrane lipids and retains its native structure and its function.

Detergents↗

PubMed2000

The NF-kappa b repressing factor is involved in basal repression and interleukin (IL)-1-induced activation of IL-8 transcription by binding to a conserved NF-kappa b-flanking sequence element.

Interleukin (IL)-8, a prototypic chemokine, is rapidly induced by the pro-inflammatory cytokine IL-1 but is barely detectable in noninduced cells. Although there is clear evidence that the transcription factor NF-kappaB plays a central role in inducible IL-8 transcription, very little is known about the cis-elements and trans-acting factors involved in silencing of the IL-8 promoter. By sequence comparison with the interferon-beta promoter, we found a negative regulatory element (NRE) in the IL-8 promoter overlapping partially with the NF-kappaB response element. Here we show that an NF-kappaB-repressing factor (NRF) binds to the IL-8 promoter NF-kappaB-NRE. Reduction of cellular NRF by expressing NRF antisense RNA results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA. Mutation of the NRE site results in loss of NRF binding and increased basal IL-8 transcription. On the other hand IL-1-induced IL-8 transcription is decreased by mutating the NRE. These data provide evidence for a dual role of the NRF in IL-8 transcription. Although in the absence of stimulation it is involved in transcriptional silencing, in IL-1-induced cells it is required for full induction of the IL-8 promoter.

Base Sequence↗