Biomedical subjects

H Offenberg

Publications and source records attributed to H Offenberg.

9 recordsLinked to original sources

PubMed2006

TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapy-induced apoptosis.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of four inhibitors of the matrix metalloproteinases, which are capable of degrading most components of the extracellular matrix. However, in recent years, TIMP-1 has been recognised as a multifunctional protein, playing a complex role in cancer. In this regard, several studies have demonstrated an antiapoptotic effect of TIMP-1 in a number of different cell types. Since chemotherapy works by inducing apoptosis in cancer cells, we raised the hypothesis that TIMP-1 promotes resistance against chemotherapeutic drugs. In order to investigate this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells and their genetically identical wild-type controls. For future studies, this cell system can be used to uncover the mechanisms and signalling pathways involved in the TIMP-1-mediated inhibition of apoptosis as well as to investigate the possibility of using TIMP-1 inhibitors to optimise the effect of conventional chemotherapy.

Animals↗

PubMed2003

Cloning and characterization of the bovine EGR4 gene and evaluation as candidate gene for bovine spinal dysmyelination.

Genes of the early growth response (EGR) family encode transcription factors with a highly conserved DNA binding zinc finger domain, which regulate a variety of genes, e.g. late myelin genes. Here, the cloning, genomic structure and expression of the bovine orthologue of the EGR4 gene are reported. The gene consists of two exons and encodes a 482 amino acid protein with a Cys2His2 zinc finger structure. The predicted protein shares between 80 and 87% identity to mouse, rat and human EGR4 proteins and all four species share almost complete identity in the DNA-binding domain. The bovine transcript is alternatively spliced by retaining intronic sequence, giving rise to two different mRNAs differing in three nucleotides and resulting in an extra alanine residue in the longer variant of the predicted protein. The gene was mapped by radiation hybrid (RH) mapping to markers on bovine chromosome 11. EGR4 transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the frontal cortex and cerebellum, and a low expression level was also detected in the liver. The EGR4 gene was evaluated as a candidate gene for bovine spinal dysmyelination (BSD). Sequencing of the gene from a homozygous affected animal and a heterozygous carrier revealed a single base mutation that leads to an amino acid substitution at residue 322 in EGR4. Genotype analysis of this polymorphism in a pedigree segregating for BSD, as well as in a panel of different cattle breeds, and sequence analysis of the entire coding region suggested that the EGR4 is not the gene responsible for BSD. Furthermore, 87 animals of different cattle breeds were screened for single-nucleotide polymorphisms (SNPs) resulting in the identification of two SNPs in EGR4.

Amino Acid Sequence↗

PubMed2000

mRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

Animals↗

PubMed2000

Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat.

Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase. They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length. Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods. It is not known at which step in meiotic recombination this association with the AEs is established. In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components. The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar. MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected. From preleptotene until early zygotene they were present throughout the nucleus. In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene. We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination. Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place.

Animals↗

PubMed1999

A high proportion of bovine blastocysts produced in vitro are mixoploid.

Fluorescence in situ hybridization with chromosome 6- and chromosome 7-specific probes was used to assess the extent of chromosome abnormalities in developing bovine blastocysts at 7-8 days after insemination in vivo or in vitro. Interphase nuclei (N = 10 946) were analyzed from 151 blastocysts produced in vitro and from 28 blastocysts recovered from superovulated animals. This revealed that 72% (109 of 151) of the in vitro-produced blastocysts were mixoploid, i.e., were a mixture of normal, diploid, and polyploid cells. However, only a small fraction of the total number of cells were chromosomally abnormal. Of the mixoploid blastocysts, 83% (91 of 109) contained less than 10% polyploid cells, 13% (14 of 109) contained 11-25% polyploid cells, and only 4% (4 of 109) of the blastocysts had more than 25% polyploid cells per blastocyst. In contrast, a significantly lower proportion (25%) of mixoploidy was found in 28 bovine blastocysts developed in vivo (p < 0.0001). All of the mixoploid blastocysts that had developed in vivo contained less than 10% polyploid cells. No entirely aneuploid blastocysts, i. e., blastocysts in which all cells had the same type of chromosome abnormality, were found in either of the groups. Taken together, the most common chromosome abnormalities observed were diploid-triploid mixoploidies and diploid-tetraploid mixoploidies. Thus, our results confirm earlier reports that morphologically normal bovine blastocysts developed in vivo are often mixoploids. We further show that in vitro-produced bovine blastocysts have a high rate of mixoploidy. Although the difference in mixoploidy rate detected in this study may not be general, it is an interesting phenomenon for further studies.

Animals↗

PubMed1988

Structure and function of vanadium-containing bromoperoxidases.

The properties of the vanadium-containing bromoperoxidases from the seaweeds Ascophyllum nodosum, Laminaria saccharina and the lichen Xanthoria parietina were studied. Upon reduction with sodium dithionite, these bromoperoxidases show EPR spectra which are typical of a vanadyl cation (VO2+). From the spectral parameters and a comparison with inorganic vanadyl complexes, we conclude that the ligand environment largely consists of oxygen donors. The data also show that the structure of the active sites in these enzymes is very similar. Since EPR spectra of vanadium(IV) bromoperoxidase are only obtained after reduction, the metal ion is present in the native enzymes in the 5+ oxidation state. All these enzymes loose their enzymic activity upon dialysis against citrate-phosphate (PO4(3-)) buffer at pH 3.8, containing EDTA. The brominating activity could be reconstituted by the addition of vanadate (VO4(3-)). The experiments suggest that vanadate is incorporated into these enzymes. In line with the EPR data, we propose a structure of the active site in which at least 4 oxygen atoms are present as donors for the central vanadium(V) ion. Since several inorganic peroxovanadium(V) complexes have been described, we suggest that the vanadium ion in bromoperoxidases serves as a binding site for H2O2. Upon subsequent binding of bromide this ion is oxidized by the peroxo-intermediate to form hypobromite. This model does not require valence state changes of the metal ion itself and indeed no changes in the EPR spectrum of reduced bromoperoxidase are observed upon addition of H2O2 or Br-. Further, bromoperoxidase reduced with a small excess of sodium dithionite is not active in the bromination reaction. The bromoperoxidases from the various sources show similarity in the amino-acid composition with a predominance of acidic amino acids. Distinct pH optima are observed in the bromination reaction catalysed by the bromoperoxidases. Despite the presence of the same prosthetic group in these enzymes with comparable vanadium ligand-field environment, the enzymic properties are very different. The specific activity as well as the Km for bromide differ greatly. Unlike the enzymes from the seaweeds A. nodosum and L. saccharina the bromoperoxidase from the lichen X. parietina is inhibited by low concentrations (1-5 mM) of nitrate. These bromoperoxidases have a remarkable resistance towards organic solvents such as methanol, ethanol and propanol.

Amino Acids↗

PubMed1986

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions -36 to +74 relative to the 3'-end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3'-termini are identical to those of 26S and 37S (pre-)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3'-processing of 5S pre-rRNA it is concluded that the maturation of 37S- and that of 5S pre-rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3'-ends of 26S rRNA and 37S pre-rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

Enhancer Elements, Genetic↗

PubMedDate not supplied

Comparison of the efficacy and safety of oxaprozin and nabumetone in the treatment of patients with osteoarthritis of the knee.

This multicenter, 6-week, double-blind, placebo-controlled, parallel-group study compared the efficacy and safety of oxaprozin 1200 mg once daily with that of nabumetone 1000 mg once daily in patients with moderate-to-severe osteoarthritis (OA) of the knee. To be eligible, patients had to experience a flare of OA within 2 weeks of discontinuing their usual OA medication (nonsteroidal anti-inflammatory drug or analgesic). Eligible patients were assessed at baseline and then randomized to receive oxaprozin (n = 109), nabumetone (n = 110), or placebo (n = 109). Efficacy assessments were performed at weeks 1, 2, 4, and 6. Primary efficacy variables included knee pain on weight bearing, knee pain on motion, and patient's and physician's global assessments of OA. Secondary efficacy variables included pain intensity, time to walk 50 feet, and duration of morning stiffness. Safety was evaluated by use of routine laboratory analyses; physical examination at screening, baseline, and week 6 (or study termination); assessment of symptoms at baseline and at each visit; and testing stools for occult blood at screening and between week 4 and the final visit. Adverse events were monitored throughout the study. Between-group differences in efficacy variables were evident by week 1. The mean change in improvement from baseline with oxaprozin compared with placebo was statistically significant in favor of oxaprozin at weeks 1, 2, 4, and 6 for all primary efficacy variables. The mean change in improvement from baseline with nabumetone compared with placebo, however, was statistically significant only at week 1 for knee pain on motion, patient's global assessment, and physician's global assessment. The mean change in improvement from baseline was statistically significant (P < or = 0.035) in favor of oxaprozin versus nabumetone at weeks 2 and 6 for all four primary efficacy variables and also at week 4 for knee pain on motion. The incidence of adverse clinical events between treatment groups was not statistically significant. However, nine oxaprozin-treated patients had asymptomatic liver enzyme elevations reported as adverse events. Four of these patients had reversible elevations of aspartate aminotransferase and alanine aminotransferase greater than three times the upper limit of normal range (P < 0.05); two of these patients were taking other medications known to induce liver enzyme abnormalities. The study showed that oxaprozin 1200 mg once daily was statistically significantly more efficacious than nabumetone 1000 mg once daily for the treatment of patients with moderate-to-severe OA of the knee. Both drugs were clinically well tolerated.

Aged↗

PubMedDate not supplied

The efficacy, tolerability, and safety of 1200 mg/d of oxaprozin and 1500 mg/d of nabumetone in the treatment of patients with osteoarthritis of the knee.

This 6-week, multicenter, double-masked, placebo-controlled study compared the efficacy, tolerability, and safety of the recommended starting dose of oxaprozin (1200 mg/d) and a 1500-mg/d dose of nabumetone in the treatment of patients with moderate-to-severe osteoarthritis (OA) of the knee. A total of 347 patients with a mean age of 61.1 years were randomized to receive oxaprozin (116 patients), nabumetone (115 patients), or placebo (116 patients). Adults of either sex who were older than 18 years of age were eligible for entry into the study, if they had had OA of the knee for at least 6 months. Efficacy variables included knee pain on weight bearing, knee pain on motion, patients' and physicians' global assessments of OA, pain intensity as measured on a visual analog scale, and time to walk 50 feet as quickly as possible. Efficacy variables were assessed at baseline and at weeks 1, 2, 4, and 6. Between-group differences in efficacy variables were evident by week 1. Mean improvements were significantly greater with oxaprozin than with placebo for all efficacy variables at all time periods, except knee pain on motion at weeks 2 and 4 and time to walk 50 feet at weeks 1, 2, and 4. Mean improvements were significantly greater with nabumetone than with placebo for all efficacy variables at all time periods, except the following: knee pain on weight bearing at weeks 2, 4 and 6; knee pain on motion at weeks 2 and 4; patients' global assessment at week 4; and pain intensity as measured on a visual analog scale at weeks 2 and 4. There were, however, no significant differences between oxaprozin and nabumetone in any of these efficacy variables. Adverse events were reported by 83 (71.6%) patients who took oxaprozin, by 80 (69.6%) patients who took nabumetone, and by 57 (49.1%) patients who took placebo. Adverse events were reported for significantly more patients taking oxaprozin or nabumetone than placebo. However, adverse events tended to be mild or moderate and rarely resulted in patients withdrawing from the study. Combined with the results of an earlier study, the results of this study showed that a 1500-mg/d dose of nabumetone, which is higher than the recommended starting dose of 1000 mg/d, is required for efficacy equivalent to that of the recommended starting dose of oxaprozin, 1200 mg/d, in relieving the symptoms of OA. Thus nabumetone may require dosage titration from the recommended starting dose. Oxaprozin and nabumetone were found to have similar tolerability profiles, as shown by adverse-event monitoring and withdrawal rates, as well as clinically similar safety profiles, as demonstrated by physical examinations, hematologic and biochemical laboratory testing, hemoccult testing, and adverse-event monitoring and symptom assessment.

Adult↗