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Biomedical subjects

G Camenisch

Publications and source records attributed to G Camenisch.

16 recordsLinked to original sources
PubMed2002

Generation and application of chicken egg-yolk antibodies.

Despite the fact that the use of chicken as immunization host brings many advantages to the production of polyclonal antibodies, the generation of egg yolk immunoglobulins (IgY) is rarely chosen. In this review, we report on the fast and efficient method for generation and affinity purification of IgY, in this case raised against the alpha-subunit of hypoxia-inducible factor-1 (HIF-1). The IgY antibody was successfully applied in a variety of methods and a number of different species for HIF-1alpha detection. In electrophoretic mobility shift assays, the IgY antibody recognized the native HIF-1 complex. The IgY antibody also detected HIF-1alpha protein on Western blots with extracts derived from human, monkey, pig, dog and mouse cell lines grown under hypoxic conditions. Immunofluorescence and immunoprecipitation experiments using the IgY antibody allowed detection and subcellular localization of HIF-1alpha in the nuclei of hypoxic cells. Chicken antibody production brings great benefit concerning the welfare of the immunized animals, due to non-invasive antibody harvesting with the added convenience of simple egg collection. An additional advantage is the fast and simple IgY isolation from egg yolk. IgY technology is a great improvement and should be considered as a good alternative to conventional polyclonal antibody production in mammals.

Animals↗
PubMed2001

Attenuation of HIF-1 DNA-binding activity limits hypoxia-inducible endothelin-1 expression.

Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1alpha is expressed ubiquitously, HIF-2alpha is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.

Base Sequence↗
PubMed2000

Efficient translation of mouse hypoxia-inducible factor-1alpha under normoxic and hypoxic conditions.

The heterodimeric hypoxia-inducible factor-1 (HIF-1), consisting of the subunits HIF-1alpha and HIF-1beta/ARNT, is a master transcriptional regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1alpha levels very rapidly increase, mostly due to protein stabilization. However, translational regulation of HIF-1alpha has not been directly analyzed so far. Mouse HIF-1alpha exists as two mRNA isoforms (termed mHIF-1alphaI.1 and mHIF-1alphaI. 2) containing structurally different 5'-termini which might modulate translation initiation. Whereas the in vitro translation efficiency of these two mRNA isoforms was about equal, the mHIF-1alphaI.2 5'-untranslated region (5'-UTR) conferred significantly higher in vivo luciferase reporter gene activity than the mHIF-1alphaI.1 5'-UTR. Similar corresponding luciferase mRNA levels indicate translational rather than transcriptional alterations. Reporter gene expression was not affected upon exposure of transiently transfected cells to hypoxia (1% oxygen). Direct assessment of translational regulation by polysomal profile analysis of HeLaS3 cells showed that HIF-1alpha (and to a lower extent ARNT) mRNA was found mainly in the translationally active polyribosomal fractions under both normoxic and hypoxic conditions. In contrast, the association of mRNAs for beta-actin and ribosomal protein L28 with the polyribosomal fractions was substantially reduced under hypoxic conditions, suggesting decreased overall protein synthesis. Thus, efficient translation of mouse HIF-1alpha in a situation where the general translation efficiency is reduced represents a prerequisite for the very rapid accumulation of HIF-1alpha protein upon exposure to hypoxia.

Animals↗
PubMed2000

Epolones induce erythropoietin expression via hypoxia-inducible factor-1 alpha activation.

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 micromol/L) potently induce HIF-1 alpha protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 micromol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 micromol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1 alpha by intracellular iron chelation. (Blood. 2000;96:1558-1565)

Animals↗
PubMed2000

A coumarin-based prodrug strategy to improve the oral absorption of RGD peptidomimetics.

In recent years, major progress has been made in the design and synthesis of fibrinogen antagonists, which are peptidomimetic Arg-Gly-Asp (RGD) analogs. These RGD analogs are very promising antiplatelet agents. However, the clinical development of orally active RGD analogs has been hindered by the low oral bioavailability of many such RGD analogs. Aimed at enhancing their oral bioavailability, we have synthesized several coumarin-based cyclic prodrugs of RGD analogs, which have the two most polar functional groups, a carboxyl and an amino group, masked as an ester and an amide, respectively. As expected, these cyclic prodrugs have higher membrane interaction potentials as estimated by determining their partitioning between aqueous buffer and an immobilized artificial membrane than the corresponding RGD analogs. Consequently, these cyclic prodrugs are 5-6-fold more able to permeate monolayers of Caco-2 cells, an in vitro cell culture model of the intestinal mucosa barrier. Preliminary studies using dog also indicate the promising potential of using this coumarin-based prodrug strategy to improve the oral bioavailability of such RGD analogs.

Animals↗
PubMed1999

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1alpha protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF-1alpha signal was weak or absent, respectively. In electrophoretic mobility shift assays, affinity-purified IgY antibody was shown to recognize the native HIF-1 (but not the related HIF-2) complex that specifically binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermore, IgY antibody immunoprecipitated HIF-1alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1alpha in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1alpha fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies.

Animals↗
PubMed1999

Oxygen tension modulates beta-globin switching in embryoid bodies.

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on beta-globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst-derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (betaH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (betamaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7-diaminofluorene-based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia-inducible factor-1 (HIF-1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered beta-globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF-1alpha isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF-1 modulates beta-globin expression during development.

Aerobiosis↗
PubMed1999

Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1alpha in hypoxia. As shown by immunofluorescence, HIF-1alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1alpha. Co-immunoprecipitation of HIF-1alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1alpha. During biochemical separation, both HIF-1alpha and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

Animals↗
PubMed1998

Up-regulation of hypoxia-inducible factor-1alpha is not sufficient for hypoxic/anoxic p53 induction.

Oxygen-deprived regions of a solid tumor can induce tumor suppressor p53 expression and hence select for p53-mutant tumor cells with diminished apoptotic potential. It has been proposed that the hypoxia-inducible factor-1 (HIF-1) alpha subunit binds to p53 and protects it from proteasomal degradation. However, we found that hypoxic conditions that strongly induce HIF-1-dependent endogenous gene expression as well as HIF-1alpha protein neither induce p53-dependent gene expression nor p53 protein. The iron chelator deferoxamine induced both HIF-1alpha and p53, but p53 up-regulation could still be detected in HIF-1alpha-deficient cells, suggesting that mechanisms other than HIF-1alpha activation contribute to oxygen-regulated p53 induction.

Animals↗
PubMed1998

Oxygen-regulated erythropoietin gene expression is dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.

Animals↗
PubMed1998

Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors.

The influence of physicochemical properties, including lipophilicity, H-bonding capacity and molecular size and shape descriptors on brain uptake has been investigated using a selection of marketed CNS and CNS-inactive drugs. It is demonstrated that the polar surface area of a drug can be used as a suitable descriptor for the drugs' H-bonding potential. A combination of a H-bonding and a molecular size descriptor, i.e., the major components of lipophilicity and permeability, avoiding knowledge of distribution coefficients, is proposed to estimate brain penetration potential of new drug candidates. Previously reported experimental surface activity data appear to be strongly correlated to molecular size of the drug compounds. Present analysis offers a modern basis for property-based design and targeting of CNS drugs.

Blood-Brain Barrier↗
PubMed1998

Estimation of permeability by passive diffusion through Caco-2 cell monolayers using the drugs' lipophilicity and molecular weight.

A recently developed, new theoretical absorption model for passive diffusion through biological membranes describing the dependency of membrane permeability on lipophilicity and molecular size, predicts different sigmoid-hyperbolic permeability-lipophilicity relationships for different molecular weight ranges. This model has been tested with experimental in vitro cultured epithelial cell (Caco-2) permeability data for structurally diverse drugs differing in lipophilicity, ionization state and molecular size. These data were pooled with literature values. Using this simple physicochemical approach, the permeability of a compound through Caco-2 cells by passive diffusion can be predicted from the compounds' distribution coefficient in 1-octanol/water (log D(oct)) and its molecular weight (MW). Deviations from this expected behaviour may point to the involvement of biological components in the transport process, which may require further investigations.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Shapes of membrane permeability-lipophilicity curves: extension of theoretical models with an aqueous pore pathway.

The objective of this study was to rationalize the shape of membrane permeability-lipophilicity curves, when considering, in addition to the usual transcellular route, a parallel diffusion pathway through aqueous pores as present in biological membranes. The theoretical influence of different pH in donor and acceptor compartment and the molecular weight on the permeability curves was studied. We combined and extended two previously proposed absorption models, namely one describing diffusion through a simple membrane (two stagnant aqueous and two organic layers in series, no pores) as the sum of the two distribution steps at both membrane interfaces, and a second theoretical model considering the sum of different diffusional resistances through stagnant layers and membrane, respectively. Under certain conditions the equivalence of the two-step distribution model and the diffusional resistance model can be demonstrated. Incorporation of an aqueous diffusion pathway leads to an extended two-step distribution model. This theoretical membrane permeation model will permit a more physicochemical-based interpretation of permeation data and shows that combined log D values and molecular weight are important determinants for membrane transport processes through, e.g. Caco-2 monolayers and the mucosal GI membranes. We have demonstrated that the well-known sigmoidal permeability-lipophilicity relationship should be considered as a molecular weight-dependent set of sigmoidal relationships.

Algorithms↗
PubMed1996

A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells.

We describe the ability of novel episomally maintained vectors to efficiently promote gene expression in embryonic stem (ES) cells as well as in established mouse cell lines. Extrachromosomal maintenance of our vectors is based on the presence of polyoma virus DNA sequences, including the origin of replication harboring a mutant enhancer (PyF101), and a modified version of the polyoma early region (LT20) encoding the large T antigen only. Reporter gene expression from such extrachromosomally replicating vectors was approximately 10-fold higher than expression from replication-incompetent control plasmids. After transfection of different ES cell lines, the polyoma virus-derived plasmid variant pMGD20neo (7.2 kb) was maintained episomally in 16% of the G418-resistant clones. No chromosomal integration of pMGD20neo vector DNA was detected in ES cells that contained episomal vector DNA even after long term passage. The vector's replication ability was not altered after insertion of up to 10 kb hprt gene fragments. Besides undifferentiated ES cells, the polyoma-based vectors were also maintained extrachromosomally in differentiating ES cells and embryoid bodies as well as in established mouse cell lines.

Animals↗
PubMed1996

Review of theoretical passive drug absorption models: historical background, recent developments and limitations.

In the drug discovery process the optimization of a promising lead to an orally bioavailable drug remains a difficult task. Recent progress in the understanding of the role of physicochemical properties in membrane permeability relevant to important processes such as drug absorption and blood-brain barrier crossing, brings rational drug delivery more within reach. In the last thirty years a number of theoretical transport and absorption models have been developed to describe mathematically how a drug is being passively transported from its site of administration to its site of action and how a compound passes a membrane. The goal of such models is to rationalize the physical significance of the observed non-linear structure-permeability relationships. The models are based on various views on the composition of the biological membranes and on the underlying diffusion and distribution mechanisms. Often simplifications reducing the mathematical complexity are made. We review here a selection of the most important models and discuss modern views on the role of lipophilicity and various pathways through membranes.

Absorption↗