PubMed Health⌕ Search

Biomedical subjects

M A Schmidt

Publications and source records attributed to M A Schmidt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Unsulfated DTPA- and DOTA-CCK analogs as specific high-affinity ligands for CCK-B receptor-expressing human and rat tissues in vitro and in vivo.

Receptors for regulatory peptides such as somatostatin or vasoactive intestinal polypeptide are expressed by a number of human neoplasms and can be visualized in vivo with peptide receptor scintigraphy. Recently, the CCK-B receptor, which binds both gastrin and cholecystokinin with high affinity, was shown using in vitro methods to be overexpressed in a number of human tumor tissues, including medullary thyroid carcinomas, small cell lung cancers, astrocytomas, gastrointestinal tumors, and stromal ovarian cancers. In the present study, we have designed novel, unsulfated CCK octapeptide analogs linked to the metal chelating DTPA and DOTA, and have tested them for their binding affinity to CCK-B receptor-positive tissue from human tumors: The most potent compounds assayed were DTPA-[Nle28, 31]-CCK(26-33) (MP2286) and DTPA-[d-Asp26,Nle28,31]-CCK(26-33) (MP2288) with an IC50 of 1.5 nM. For comparison, analogs with C-terminal DTPA, such as [Nle28,31,Aphe33(p-NH-DTPA)]-CCK(26-33) and CCK-(26-33)-NH(CH2)2 NH-DTPA, had an IC50 of >100 nM. DOTA-[D-Asp26, Nle28,31]-CCK(26-33) had an IC50 of 3.9 nM. The compounds were selective for CCK-B receptors as they did not bind with high affinity to CCK-A receptors expressed in human tumors (meningiomas or gastroenteropancreatic tumors). In vivo rat biodistribution studies with indium-111 labeled MP2286 and MP2288 showed that the primary mode of clearance was renal, and the primary sites of uptake (% ID/g 24 h p.i.) were kidneys (0.270 and 0.262, respectively) and the gastrointestinal tract. The CCK-B receptor-expressing gastric mucosa showed specific in vivo accumulation of 111In-labeled MP2288 which could be blocked in the presence of excess unlabeled MP2288. 111In-labeled MP2286 and MP2288 were also found to be stable in human plasma whereas both compounds were degraded in urine (>40% after 3 h at 37 degrees C). The affinity, specificity, biodistribution, and stability of these two DTPA-CCK analogs indicate that these compounds have substantial promise for use in the in vivo visualization of CCK-B receptor-expressing tumors.

Animals↗
PubMed1998

Diffusely adhering Escherichia coli strains induce attaching and effacing phenotypes and secrete homologs of Esp proteins.

Recent epidemiological studies indicate that Escherichia coli strains which exhibit the diffuse-adherence phenotype (DAEC strains) represent a potential cause of diarrhea in infants. We investigated the interaction of DAEC strains isolated from diarrhea patients in Brazil and in Germany with epithelial cells in tissue culture. The investigated strains were identified as DAEC strains by (i) their attachment pattern, (ii) presence of genes associated with the Dr family of adhesins, and (iii) lack of genetic markers for other diarrhea-associated E. coli categories. Several clinical DAEC isolates were shown to secrete similar patterns of proteins into tissue culture medium. Protein secretion was found to be regulated by environmental parameters, namely, medium, temperature, pH, and iron concentration. DAEC strains secreting these proteins induced accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures. These changes were phenotypically similar to the attaching and effacing (A/E) lesions observed with enteropathogenic and some enterohemorrhagic E. coli strains carrying the locus of enterocyte effacement (LEE) pathogenicity island. Proteins homologous to the EspA, EspB, and EspD proteins, necessary for signal transduction events inducing A/E lesions, were identified by sequence analysis and cross-reaction of specific antibodies. However, initially nonadhering strains secreting these proteins induced signal transduction events only after prolonged infection. These results indicate that secretion of the Esp proteins alone is not sufficient for efficient signal transduction. This study further shows that some DAEC strains are likely to contain a homolog(s) of the LEE locus which may contribute to the pathogenic potential of DAEC.

Actins↗
PubMed1998

Human intestinal M cells exhibit enterocyte-like intermediate filaments.

BACKGROUND: The derivation and ultrastructural composition of M cells covering the lymphoid follicles of Peyer's patches is still unknown. Results from different animal models have shown that there are species specific differences in the composition of intermediate filaments between M cells and neighbouring enterocytes. Little is known, however, about intermediate filaments of human M cells. AIMS: To compare components of the cytoskeleton of human M cells with those of adjacent absorptive enterocytes. METHODS: The expression and localisation of different cytokeratins, vimentin, and desmin in M cells was determined on follicle associated epithelia of human appendix using immunohistochemistry and immunogold electron microscopy. RESULTS: Cytokeratins specific for human intestinal epithelial cells such as cytokeratins 8, 18, 19, and 20 were expressed in both absorptive enterocytes and M cells with no differences in intensity and cellular distribution between both cell types. Vimentin and desmin, tissue specific markers of either mesenchymal or myogenic cells, as well as other cytokeratins were not detectable in enterocytes or M cells. CONCLUSION: This is the first study on the structure of intermediate filaments in human intestinal M cells. Our results show that in contrast to several animal models, human M cells apparently do not differ from adjacent enterocytes in the composition of their intermediate filament cytoskeleton. The presence of enterocyte like cytokeratins and the absence of other cytokeratins as well as of vimentin and desmin supports the hypothesis of an epithelial origin of human intestinal M cells and suggests that M cells may derive from differentiated enterocytes.

Adult↗
PubMed1997

Membrane anchored synthetic peptides as a tool for structure-function analysis of pertussis toxin and its target proteins.

Solid phase fixed peptides are useful tools for the investigation of enzyme substrate interactions. We could show that interactions of target sequences, like e.g. the acceptor domain of G-proteins for the of mono ADP-ribosylation by pertussis toxin and the toxin itself can be studied by this method. Reaction specificity is identical to modification of free peptides. The method is extremely versatile and has enormous potential if the focus is on the effects of substrate modification or substrate length.

Adenosine Diphosphate↗
PubMed1997

Non-breath-hold lung magnetic resonance imaging with real-time navigation.

Magnetic resonance imaging (MRI) with navigating techniques based on consecutive breath-holds demand a level of respiratory control that is often beyond the capability of patients with lung disease. The objectives of this investigation were to develop and evaluate a navigating technique for lung MRI that does not rely on patient cooperation. Navigating techniques were implemented at 0.5 T using conventional imaging techniques of short echo-time and imaging during normal breathing in the diastolic phase of the cardiac cycle. A column of spins, orthogonal to the diaphragm, was excited both immediately before and after the imaging segment. These signals were processed in real time to provide the position of the lung-diaphragm interface. An imaging segment was considered correctly acquired only when the interface position was within the acceptance window both before and after the acquisition of the segment. A distribution of lung-diaphragm interface positions obtained during normal respiration was employed to define the acceptance window. In the case of multislice techniques, the position of the lung-diaphragm interface immediately before the imaging segment was also employed to decide which phase-encoding step to acquire next, therefore reducing the apparent frequency of the respiratory motion. A distribution of interface positions, updated in real time, served as a reference for the allocation of phase-encoding steps according to diaphragm position. The lung images obtained represent a significant advance in image quality, improving further the ability of MR to detect and monitor pulmonary disease. Motion artifacts were reduced, and images reliably demonstrated smaller vessels, which are not normally visible without navigation.

Computer Systems↗
PubMed1997

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes.

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides GM1, GM3, GD1a, GD1b, GT1b and asialo-GM1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for GM1 (Ka = 1.8.10(8)M-1), a lower one for asialo-GM1 (Ka = 1.0.10(7)M-1) and no affinity for GM3. The C-fragment of tetanus toxin binds to ganglioside GD1a, GD1b and GT1b containing membranes with similar affinity (Ka approximately 10(6)M-1), while no binding was observed with GM3. Pertussis toxin binds to membranes containing the ganglioside GD1a with a binding constant of Ka = 1.6.10(6)M-1, but only if large amounts (40 mol%) of GD1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to GD1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to GD1a.

Cholera Toxin↗
PubMed1997

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B.

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.

Animals↗
PubMed1997

Endocytosis and retrograde transport of pertussis toxin to the Golgi complex as a prerequisite for cellular intoxication.

The uptake mechanism of pertussis toxin (PT) in CHO and insulin-producing HIT-T15 cells was studied. By electron microscopy after direct labeling of the toxin with gold particles, PT was found to be taken up by receptor-mediated endocytosis. The presence of active pertussis toxin in the Golgi complex was shown by subcellular fractionation. The importance of the Golgi localization of pertussis toxin for the S1-dependent ADP-ribosylation of G-proteins was investigated employing Brefeldin A (BFA) treatment to disrupt Golgi structures. Treatment with Brefeldin A completely blocked the pertussis toxin mediated ADP-ribosylation of cellular G-proteins in CHO and HIT-T15 cells, whereas the BFA-resistant MDCK cells were not protected. A mutant CHO cell line (V24.1) exhibiting a temperature-sensitive Golgi complex could be protected when grown at restrictive conditions. These results strongly indicate that retrograde transport to the Golgi network is a necessary prerequisite for pertussis toxin mediated ADP-ribosylation of G-proteins and thus also for cellular intoxication.

Adenosine Diphosphate Ribose↗
PubMed1997

Pertussis toxin. Entry into cells and enzymatic activity.

In this study we present data supporting the concept of a retrograde transport mechanism for pertussis toxin to the Golgi complex. We also describe a novel GTP-binding 32 kDa cellular target protein, which is ADP-ribosylated upon exposure to PT and different from the classical PT substrate, the alpha-subunit of Gi proteins.

Adenosine Diphosphate Ribose↗
PubMed1997

Towards targeting strategies for oral immunization--identification of marker antigens in rat M cells.

Specialized M cells of the follicle-associated epithelium (FAE) of Peyer's patches in the gastrointestinal and respiratory tracts play a crucial role in the immune surveillance of mucosal surfaces and are essential for the induction of mucosal immune responses. These cells transport luminal antigens to the underlying germinal centers and thereby initiate an immune response or induce tolerance. Mucosal immune responses could be significantly enhanced if oral antigens could be directly targeted to the apical surface of M cells. However, thus far M cells have not been isolated and suitable surface markers have not been described. For the characterization and identification of potential molecular markers of M cells the follicle associated epithelium of Peyer's patches from the small intestine of rats was analyzed by indirect immunofluorescence and immunogold electronmicroscopy. The expression of intermediate filament proteins in the FAE was found to be heterogeneous. Immunoreactivity of a specific monoclonal cytokeratin 8 antibody (clone 4.1.18) was selectively found in cells which were additionally characterized by the lack of staining for alkaline phosphatase in their apical membranes. In the FAE this property is an accepted criterium for the presence of M cells. The antigen recognized by the clone 4.1.18 is expressed in substantially higher amounts in M cells of Peyer's patches as compared to neighbouring epithelial cells or "normal" enterocytes and thus can be employed as an intracellular molecular marker for M cells of Peyer's patches in rats. It is expected, that this marker will be very helpful for the isolation of M cells.

Administration, Oral↗
PubMed1996

Quantification of immunological membrane reactions employing a digital desk top scanner and standard graphics software.

Many common techniques used in biochemical and molecular biology analysis involve the generation of signals by immunological or hybridization detection reactions on membranes or photographic films (e.g. 'windrose blots', immunoblots). The possibility to undertake comparative and quantitative evaluation of these signals is a basic requirement in most applications. However, visual inspection of immunological detection by colour reactions or by autoradiography using a photographic film does not usually provide a reliable basis for the quantification and interpretation of results. Thus, there is a need for a reproducible, (semi-)quantitative method with an option for numeric read-out and comparable graphic display. Here, we describe a simple method for the reliable and reproducible evaluation of signals generated on membranes or photographic films which combines standard graphics software with a low-budget desk top scanner.

Autoradiography↗
PubMed1996

The safety of calcium channel blockers in human pregnancy: a prospective, multicenter cohort study.

OBJECTIVE: Our purpose was to examine the potential teratogenicity of calcium channel blockers. STUDY DESIGN: Six teratogen information services prospectively collected and followed up 78 women with first-trimester exposure to calcium channel blockers. Pregnancy outcome was compared (by paired t text of chi2 analysis) with that of a control group matched for maternal age and smoking. RESULTS: There was no increase in major malformation (2/66=3.0% [calcium channel blockers] vs 0% [nonteratogenic controls], p=0.27); a fivefold increase was ruled out (baseline 2%, alpha = 0.05, beta = 0.20). The defects reported were attributable to maternal diabetes or coingestion of teratogens. The increase in preterm delivery 28% [calcium channel blockers] vs 9% [nonteratogenic controls], p=0.003), attributed to maternal disease by stepwise regression, was the most important factor responsible for the observed decrease in birth weight (mean -334 gm vs nonteratogenic controls, p=0.08). CONCLUSION: This study suggests that calcium channel blockers do not represent a major teratogenic risk.

Abnormalities, Drug-Induced↗
PubMed1996

Processing of the AIDA-I precursor: removal of AIDAc and evidence for the outer membrane anchoring as a beta-barrel structure.

The AIDA-I adhesin known to be responsible for the diffuse adherence (DA) phenotype of the diarrhoeagenic Escherichia coli (DAEC) strain 2787 has been shown previously to be synthesized as a precursor protein and to undergo additional C-terminal processing. Here, the C-terminal processing of the AIDA-I precursor and the outer membrane topology of the cleaved C-terminal fragment, AIDAc, were investigated. By isolation of the cleaved AIDAc fragment and N-terminal sequencing, the C-terminal cleavage site was identified between Ser-846 and Ala-847 thereby indicating a molecular mass of 47.5 kDa for AIDAc. The correct processing to AIDA-I and AIDAc in OmpT, OmpP and DegP protease-deficient E. coli strains as well as in avirulent salmonellae and shigellae points to an autocatalytic cleavage mechanism. The cleaved AIDAc was localized in the outer membrane. A leader sequence-AIDAc fusion was efficiently routed to the outer membrane. Analysis by protease digestion, secondary-structure prediction and modelling, by comparison with structurally related bacterial proteins like the IgA1 protease from neisseria, the vacuolating toxin from Helicobacter pylori, and the VirG protein of Shigella flexneri, strongly indicates that AIDAc is present in the outer membrane as a beta-barrel structure.

Adhesins, Escherichia coli↗
PubMed1996

Immunocytochemical characterization of the follicle-associated epithelium of Peyer's patches: anti-cytokeratin 8 antibody (clone 4.1.18) as a molecular marker for rat M cells.

For the characterization and identification of potential molecular markers of M cells, the follicle-associated epithelium (FAE) of Peyer's patches from the small intestine of rats was analyzed by indirect immunofluorescence and immunogold electron microscopy. The expression of intermediate filament proteins in the FAE was found to be heterogeneous. Immunoreactivity of a specific monoclonal cytokeratin 8 antibody (clone 4.1.18) was selectively found in cells which were additionally characterized by the lack of staining for alkaline phosphatase in their apical membranes. In the FAE, this property is an accepted criterium for the presence of M cells. Further, these cells were found to be devoid of mucin and were thus distinct from goblet cells. The antigen recognized by the clone 4.1.18 is expressed in substantially higher amounts in M cells of Peyer's patches as compared to neighboring epithelial cells or "normal" enterocytes and thus can be employed as an intracellular molecular marker for M cells of Peyer's patches in rats.

Animals↗
PubMed1995

Identification of binding proteins for pertussis toxin on pancreatic beta cell-derived insulin-secreting cells.

The ability of pertussis toxin (PT) to recognize and bind to surface proteins on cells derived from pancreatic insulin-secreting beta cells and alpha cell-like glucagon-producing cells was investigated employing HIT-T15 (beta cell-derived) and In-R1-G9 (alpha cell-like) cell lines. PT recognition of membrane binding proteins on HIT-T15 and In-R1-G9 cells was first assessed with immunofluorescence microscopy in tissue culture. Both cell lines were equally well recognized by PT. N-octylglucoside extracts of whole cells and isolated membranes were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. PT, the B-oligomer, or the isolated PT dimers S2-S4 and S3-S4 recognized distinct proteins in HIT-T15 and In-R1-G9 cells of about 220 kDa. Recognition by the sialic acid specific Sambucus nigrica lectin identified these proteins as sialoglycoproteins. Incubation of the blotted membrane proteins with sialidase or pretreatment of PT with anti-PT polyclonal antibodies abolished the recognition and binding of these proteins by PT. To demonstrate that these glycoproteins are also able to transduce PT mediated effects and thus might serve as PT binding proteins, the stimulation of insulin secretion in HIT-T15 cells was assessed. As the secretion of insulin in HIT-T15 cells increased about 30% upon interaction with PT it was concluded that these glycoproteins are indeed functional as PT receptors.

Animals↗
PubMed1995

Mapping of functional regions on the transferrin-binding protein (TfbA) of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae can use porcine transferrin as the sole source of iron. Two proteins with molecular masses of approximately 60 kDa (TfbA) and 110 kDa have been shown to specifically bind porcine transferrin; from the TfbA protein, three isoforms from A. pleuropneumoniae serotypes 1, 5, and 7 have been identified and characterized by nucleotide sequence analysis. Here we defined the transferrin-binding region(s) of the TfbA protein of A. pleuropneumoniae serotype 7 by TnphoA mutagenesis, random mutagenesis, and peptide spot synthesis. The amino-terminal half of the TfbA molecule, which has only 36% amino acid sequence identity among the three isoforms, was shown to be responsible for transferrin binding by TnphoA mutagenesis. This result was confirmed by analysis of six random mutants with decreased transferrin binding affinity. The subsequent analysis of overlapping 16-mer peptides comprising the amino-terminal half of the TfbA molecule revealed three domains of 13 or 14 amino acids in length with transferrin-binding activity. They overlapped, or were very close to, point mutations decreasing transferrin-binding ability. The first and third domains were unique to the TfbA protein of A. pleuropneumoniae serotype 7. In contrast, the sequence of the second domain was present in almost identical forms (12 of 14 residues) in the TfbA proteins of A. pleuropneumoniae serotypes 1 and 5; in addition, a sequence consisting of functionally homologous amino acids was present in the otherwise completely distinct small transferrin-binding proteins of Neisseria gonorrhoeae (TbpB), N. meningitidis (Tbp2), and Haemophilus influenzae (Tbp2).

Actinobacillus pleuropneumoniae↗