Detecting estrogens in food and the environment.
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Animals↗
Biomedical subjects
M Seifert
Publications and source records attributed to M Seifert.
Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function.
We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.
Creatine Kinase↗
In vitro analysis of xenoestrogens by enzyme linked receptor assays (ELRA).
Receptor binding assays are a modern approach to the effects-related analysis of xenoendocrines. Human estrogen receptors are used to develop an analytical tool for the detection of estrogenic substances in environmental samples. A sensitive Enzyme Linked Receptor Assay (ELRA) was developed in a microwell plate format. The receptor assay is based on similar principles as competitive immunoassays (ELISA). However, receptor binding always implies a biological effect, either agonistic or antagonistic. The choice of suitable tracers or hapten conjugates is an important step in assay development. For this purpose and for the determination of receptor-affinities of relevant xenoestrogens, a surface plasmon resonance (SPR) biosensor (BIAcore) was used for binding studies with immobilized receptors. Results with commercially available hapten-conjugates (estradiol-BSA) show a direct correlation between the amount of immobilized estradiol receptor and the amount of bound hapten-conjugate. Based on these BIAcore experiments an ELRA was developed. The calibration curves show a detection limit of 0.1 microgram/l for 17 beta-estradiol. Cross-reactivities of different steroids and xenoestrogens are reported.
Biosensing Techniques↗
Evidence that tamoxifen preserves bone density in late postmenopausal women with breast cancer.
Tamoxifen, which is used for treating breast cancer, exhibits estrogenic and antiestrogenic characteristics, depending on the tissue. In the human breast it acts as an antiestrogen, whereas estrogenic effects have been reported on endometrium and bone. The purpose of this study was to determine whether tamoxifen (TAM) prevents bone loss in elderly, postmenopausal women. Bone mineral density of the lumbar spine (SBD) was measured in elderly women (at least 10 years after menopause) 5 years after stage I or II breast cancer (n = 111). The results showed that SBD in untreated patients (n = 74) was significantly lower (p < 0.05) than SBD in patients (n = 37) treated with TAM over 5 years. In a subgroup of patients (n = 24) with positive estrogen receptor status, changes in SBD 12 months after discontinuation of 5-year TAM therapy were measured and compared with the changes of extended TAM treatment over a sixth year. Twelve months after withdrawal of 5-year TAM medication (n = 11) bone density decreased significantly (- 4.8+/-2.5%; p > 0.05), whereas in the group of women (n = 13) receiving extended TAM medication (20 mg) for an additional 12 months, SBD ( + 1.9+/-3.5 %) was maintained during the observation period, and was significantly higher when compared with the group of untreated patients (p <0.05). We conclude that tamoxifen has a preventive effect on trabecular bone loss at the lumbar spine, when compared to age-matched data and to untreated women with breast cancer in the late menopause. Our data give evidence of benefits to bone density provided by prolonged administration in patients after breast cancer and at risk of osteoporosis.
Aged↗
[Oral contraceptives and breast cancer--a causal relationship?].
Concern about the relationship between oral contraceptives, which have been used by more than 200 million women, and the risk of breast cancer has been present since three decades. Early studies showed no consistent results, but some authors are concerned about several subgroups having possibly an increased risk of developing breast cancer. A meta-analysis representing 90% of the data available on the topic showed a small but significant increase in the relative risk of developing breast cancer in women currently using oral contraception (1.24). This risk decreases after oral contraceptives have been ceased (1.16-1.07) and is comparable after 10 years to that of controls.
Adolescent↗
Vascular endothelial growth factor (VEGF) in human breast cancer: correlation with disease-free survival.
Studies have shown that microvessel density influences breast-cancer prognosis. Since tumor angiogenesis is considered to be substantially affected by the excretion of vascular endothelial growth factor (VEGF) from tumor cells, we examined whether VEGF concentration is different in malignant and in non-malignant breast tissue. It was also of interest to discover whether intratumoral VEGF concentration influences disease-free survival (DFS) of breast-cancer patients. Analysis is based on 120 tissue specimens taken from breast fibromas (n = 23), normal epithelial breast tissue adjacent to fibromas (n = 8) and invasive breast cancer (n = 89). VEGF concentration was quantified by using an immunoassay. Microvessel density was determined by immunostaining for factor-VIII-related antigen. Median VEGF concentration is given in pg/mg protein (25%-quantile-75%-quantile) and it was 0 (0-1.8) in normal breast tissue, 9.8 (0.52-43.0) in fibromas and 130.4 (50.8-362.2) in invasive carcinomas. A univariate Cox model revealed that node status, tumor size, estrogen-receptor concentration, histological grading and microvessel density were prognostic factors for disease-free survival in breast cancer. We found a significant correlation between VEGF concentration and microvessel count, but VEGF concentration did not significantly influence disease-free survival. Although VEGF protein was found at a significantly higher concentration in malignant than in non-malignant tissue, determination of intratumoral VEGF protein by an enzyme immunoassay was not prognostically relevant in our patient population.
Analysis of Variance↗
The three human trefoil genes TFF1, TFF2, and TFF3 are located within a region of 55 kb on chromosome 21q22.3.
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Base Sequence↗
Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis.
The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5' to nm23-H1 and another 3' to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed.
Aged↗
Mammography in women undergoing hormone replacement therapy. Possible effects revealed at routine examination.
PURPOSE: A retrospective study of screen-film mammographs was performed in 960 postmenopausal women to evaluate the possible effects of hormonal replacement therapy on the breast parenchymal pattern. MATERIAL AND METHODS: Screen-film mammographs of 754 women (mean age 51.4 years) who had undergone hormonal replacement therapy (estrogen, gestagen; mean duration 2.4 years) were compared with findings in 206 women who had not received hormonal replacement therapy (mean age 52.3 years). Mastopathic changes were analyzed according to a 4-grade scale and the incidence of circumscribed lesions (such as fibroadenomas, cysts and malignancies) was further evaluated. RESULTS: In the treated women we found moderate mastopathic changes in 47.2%, intermediate changes in 2.4%, and extensive proliferations in 0.7%. For the untreated women the corresponding figures were 33.5%, 4.8%, and 1.4%. In the treated patients fibroadenomas were apparent in 3.9%, cystic lesions in 3.3%, and malignant lesions in 1.4%. In the untreated women fibroadenomas were evident in 11.6%, cystic lesions in 7.2%, and malignant lesions in 2.4%. CONCLUSION: Mastopathic changes might increase under hormonal replacement therapy. However, the higher incidence of circumscribed lesions in the untreated women might be due to the higher number of symptomatic women who sought examination. We also speculate as to whether the increased density of the breast parenchyma in the treated women might cause a higher number of mammographically undedectable lesions.
Breast↗
Differentiation of benign and malignant breast lesions: MR imaging versus Tc-99m sestamibi scintimammography.
PURPOSE: To compare the accuracies of magnetic resonance (MR) imaging and scintimammography in differentiating benign from malignant breast lesions. MATERIALS AND METHODS: MR imaging was performed in 66 women with 75 lesions during intravenous administration of gadopentetate dimeglumine. Planar and single photon emission computed tomographic (SPECT) scintimammography were performed (with 740 MBq technetium-99m sestamibi administered intravenously) in all 66 patients with 75 lesions and in 64 patients with 73 lesions, respectively. MR imaging and scintimammographic studies were independently evaluated by using signal intensity measurements versus time or focal tracer uptake to differentiate benign from malignant lesions. Histopathologic proof was obtained in 63 lesions. Twelve lesions were monitored with follow-up. RESULTS: MR imaging was false-negative in one and false-positive in nine lesions. Planar scintimammography was false-negative in 10 and false-positive in six lesions. SPECT scintimammography was false-negative in four and false-positive in 10 lesions. Sensitivities and specificities for malignancy were, respectively, 96% and 82% for MR imaging, 62% and 88% for planar scintimammography, and 83% and 80% for SPECT scintimammography. CONCLUSION: Both MR imaging and scintimammography are useful in the evaluation of breast cancer. MR imaging is more sensitive and as specific as scintimammography.
Adult↗
[Laparoscopic colon surgery in the regional hospital: initial experiences after 65 interventions].
321 colorectal operations have been performed in our institution in a 4-year period from 1992 to 1996. They included 65 laparoscopic interventions for benign disease or for palliative treatment of malignancy. 40 patients underwent low anterior resections, mostly for diverticular disease. 11 right hemicolectomies, 3 left hemicolectomies, 1 total colectomy and 7 rectopexies (6 with sigmoid resection) were performed. 3 patients with advanced malignant disease underwent colostomy. There was a conversion rate of 26%, largely due to adhesions or unclear anatomy, decreasing with increasing experience. One trokar-injury of a major vessel required immediate conversion to laparotomy. 2 reoperations were performed, for an anastomotic leakage after low anterior resection and an incarcerated port-site-hernia respectively. A rather high rate of wound infections (27%) was reduced to 15% by modifying the technique for retrieval of the specimen. The median postoperative hospital stay was 11.6 days. The trend towards shorter hospital stay and recovery should not result in uncritical use of laparoscopy in colon surgery, in an attempt to meet the rising demand for less postoperative pain and shorter hospital stay. This criticism mainly applies to tumor surgery, where laparoscopic procedures are indicated in our institution only for palliative treatment.
Adult↗
Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS).
Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria. The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS). Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specificity of antigen antibody interactions. Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties. In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB03 used was screened for lipid A/LPS reactivity in ELISA experiments. The binding was confirmed in the described blot system and depends on the membranes and blocking agents used. The use of nitrocellulose versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining.
Animals↗
Toward optimal laboratory use. Problems in laboratory testing in primary care.
OBJECTIVE: To examine the frequency and characteristics of problems in laboratory testing in primary care physicians's offices and their impact on health care. DESIGN: Prospective descriptive study in which participating office-based primary care clinicians reported each occurrence of any laboratory incident during a 6-month study. Each identified problem was reported on a structured data collection instrument with an open-ended description of the problem. SETTING: Primary care physicians' offices in North America. PARTICIPANTS: One hundred twenty-four primary care clinicians in 49 practices of the Ambulatory Sentinel Practice Network (ASPN). MAIN RESULTS: A total of 180 problems were reported, yielding a crude rate of 1.1 problems per 1000 patient visits. Problems involving test ordering and specimen handling were the most common (56%), while those attributable to the test analysis itself accounted for 13% of the total. In the judgment of the practice staff, 27% of the reported problems had an impact on patient care. Of the 24 cases for which the specific impact was known and reported, half of the impacts were minor and about half were significant, as judged by whether or not the diagnosis and/or treatment of the patient was measurably affected. CONCLUSIONS: Clinically apparent problems with laboratory testing in primary care were found at a rate of 1.1 problems per 1000 patient visits. Of the laboratory problems that occurred in this study, 27% were judged by the physician to have an effect on patient care.
Clinical Laboratory Techniques↗
Homocysteine modification of HLA antigens and its immunological consequences.
Homocysteine-treated cells can be specifically lysed by cytotoxic T lymphocytes (CTL) identifiable in patients with ankylosing spondylitis and reactive arthritis. Sensitization of target cells involves disulfide bonding and the interaction between homocysteine and HLA antigens occurs in a pre-Golgi compartment in the cells. Salmonella-infected B cells are also lysed by homocysteine-specific CTL, suggesting that intracellular invading microorganisms may provide homocysteine which would gain access to the newly synthesized intracellular HLA molecules and modify them inside the cells. Two different mechanisms for homocysteine modification of HLA antigens are proposed: homocysteine could bind directly to the unpaired cysteine residues in HLA antigens, or it could bind indirectly to HLA antigens through cysteine-containing peptides bound to them. Thus, HLA antigens containing unpaired cysteine residues (e.g. HLA B27) could be modified by homocysteine directly or indirectly, while HLA antigens without unpaired cysteine residues (e.g. HLA A68) could only be modified indirectly. The results are discussed in relation to the potential involvement of homocysteine-specific CTL in ankylosing spondylitis and reactive arthritis, both of which are related to bacterial infections, associated with HLA B27, and considered to be autoimmune diseases.
Adult↗
Mapping protein-protein contact sites using cellulose-bound peptide scans.
We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). In addition, the interaction between TNF-alpha and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-alpha, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-alpha, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-beta in the TNF-beta-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.
Amino Acid Sequence↗
Neutralizing murine monoclonal anti-interleukin-10 antibodies enhance binding of antibodies against a different epitope.
Fifteen murine hybridoma lines that produce monoclonal antibodies against human interleukin-10 (IL-10), which is one of the most important regulatory cytokines of the immune system, were raised. Twelve of these antibodies, all in the class IgG1/kappa, recognize three groups of epitopes: A, B and C. All antibodies in these groups inhibit binding of antibodies of the same group to IL-10 and none of them inhibit binding of an antibody of another group. Two IgM/kappa antibodies and one IgG1/kappa antibody, with low affinity, have distinct binding properties and cannot be assigned to one of these groups. Antibodies from all three epitope groups inhibit the biological activity of IL-10. The three antibodies of group A neutralize IL-10 in an approximately equimolar ratio, at concentrations as low as 10 pM. In addition to their high neutralizing capacity, antibodies of group A enhance the binding of antibodies of group C to IL-10. The on-rate of binding of the two antibodies CB/RS/10 and 11 (group C) increased five- to seven-fold, when one of the antibodies CB/RS/1, 2 or 14 (group A) is bound to IL-10.
Adjuvants, Immunologic↗
Generation and characterization of a human monoclonal IgM antibody that recognizes a conserved epitope shared by lipopolysaccharides of different gram-negative bacteria.
A hybridoma cell line secreting a human monoclonal antibody (humab) directed to an epitope in the lipid A region of lipopolysaccharides of Gram-negative bacteria was isolated. Peripheral blood lymphocytes (PBL) obtained from a healthy volunteer were immortalized by Epstein-Barr virus (EBV) transformation. Lymphoblastoid cell lines (LCL) secreting antibodies to the lipopolysaccharides of Gram-negative bacteria were determined by an enzyme-linked immunosorbent assay (ELISA) and subsequently fused with the human-mouse heteromyeloma cell line CB-F7 by polyethylenglycol (PEG)-mediated fusion. A hybridoma line producing a humab (LPD5H4), of the IgM/lambda isotype, which strongly reacted with the lipid A portion of Salmonella and E. coli spp. in ELISA, was established. The antibody was purified by hydrophobic interaction chromatography and gel filtration. Immunoblotting experiments showed a strong reactivity of the humab LPD5H4 with the lower molecular species of different rough and smooth lipopolysaccharide (LPS) types of the bacteria species Salmonella, E. coli, Klebsiella, and Neisseria meningitidis, whereas those of Pseudomonas spp. were negative. Binding of humab LPD5H4 to solid phase bound lipid A and different rough mutants of LPS could be inhibited by the corresponding antigens in solution. Competition assays with a murine monoclonal antibody to lipid A and with polymyxin B indicate that humab LPD5H4 recognizes its epitope in this extremely conserved part of the LPS molecule. In vitro tests demonstrated that the MAb is able to partially inhibit the LPS-induced release of TNF-alpha using isolated peripheral blood mononuclear cells (PBMC).
Animals↗
Characterization of the human nm23-H2 promoter region and localization of the microsatellite D17S396.
The transcription of human nm23 genes (nm23-H1, nm23-H2) is involved in suppression of tumor metastasis or tumor progression. Therefore the characterization of transcriptional regulatory mechanisms for both nm23 genes is very important. In this study we have isolated and analyzed the 5'-flanking region of the human nm23-H2 gene and estimated the distance to 4 kb between nm23-H2 and nm23-H1 genes. We localized the known microsatellite D17S396 within this region. Furthermore the identification of possible binding sites for MYC proteins and additionally the NM23-H2 protein itself (the transcription factor PuF for c-myc gene activation) is of importance with respect to possible para- and autoregulatory interactions. A comparison of the promoter sequences of both human nm23 genes revealed no significant sequence homology.
Animals↗