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Biomedical subjects

M A Schmidt

Publications and source records attributed to M A Schmidt.

At least 19 recordsLinked to original sources
PubMed1992

AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule.

The adherence mechanisms of enteropathogenic Escherichia coli (EPEC) to epithelial cells are still not understood. To study the molecular basis of the diffuse adherence (DA) phenotype exhibited by diarrhoeagenic E. coli expressing classical EPEC serotypes we investigated strain 2787 (O126:H27) isolated from a case of infantile diarrhoea. A 6.0 kb plasmid-derived DNA fragment mediates the DA phenotype and encodes the 100 kDa adhesin protein AIDA-I (adhesin involved in diffuse adherence). Sequencing of the entire fragment revealed two open reading frames which encoded proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as an AIDA-I precursor protein. After cleavage of the signal sequence further processing at the C-terminus of the 132 kDa precursor leads to the mature approximately 100 kDa AIDA-I. While the exact function of the cytoplasmic 45 kDa protein is not known, preliminary evidence indicates that it is necessary for the correct maturation of AIDA-I. The AIDA-I precursor exhibits significant homology with the virG(icsA) protein of Shigella flexneri which seems to be involved in the intercellular spread of invasive Shigella organisms.

Adhesins, Escherichia coli↗
PubMed1992

Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27).

The adherence of diarrhea-associated Escherichia coli to the small-bowel mucosa is an important step in the pathogenesis of diarrheal diseases. In tissue culture systems, diarrhea-associated strains show three distinct patterns of adherence: localized adherence, diffuse adherence (DA), and the recently described aggregative adherence. To study the molecular basis of the DA phenotype, we investigated the diarrhea-associated DA strain 2787 (O126:H27), isolated from a case of infantile diarrhea. The DA phenotype is mediated by a 6.0-kb DNA fragment derived from a 100-kb plasmid harbored by the wild-type strain. This fragment codes for a 100-kDa protein which can be released from the bacterial cell into the supernatant by mild heat treatment. Recombinant DA+ strains as well as the isolated 100-kDa protein were used to engender specific antisera in rabbits. As demonstrated by Western blotting (immunoblotting), the antibodies engendered by the recombinant DA+ strain recognized a 100-kDa protein in the wild-type strain 2787 and in all recombinant strains showing DA. Immunogold electron microscopy localized the 100-kDa protein to the bacterial cell surface. Serologically related proteins of similar size were detected by Western blotting in other DA+ diarrhea-associated strains belonging to enteropathogenic E. coli serotypes. The 100-kDa protein denoted AIDA-I (adhesin involved in diffuse adherence) binds in a saturable fashion to HeLa cells. AIDA-I-specific immunoglobulin G antibodies--and, to an even greater extent, Fab fragments derived thereof--inhibited bacterial attachment to HeLa cells. This is direct evidence that the 100-kDa protein is the adhesin mediating the DA phenotype of these diarrhea-associated strains and is representative of a group of serologically related proteins in other DA+ strains.

Bacterial Adhesion↗
PubMed1991

Characterization of monoclonal antibodies directed against domains of pertussis toxin involved in receptor recognition.

The exotoxin pertussis toxin (PT) produced by virulent Bordetella pertussis bacteria is regarded as the main virulence factor of the organism and held responsible for most of its pathological effects. Identification of functional sites on PT would greatly facilitate site-specific detoxification and thus also the development of a new vaccine. For the investigation of structure-function aspects of PT we have prepared and characterized eleven monoclonal antibodies (mAbs) (UB-A1, UB-A2, UB-A10, UB-B7, UB-B12, UB-D4, UB-D7, UB-D10, UB-F7, UB-G1, and UB-G12) directed at the native toxin. Only UB-B12 and UB-D10 recognized PT in Western blotting indicating that most of the mAbs were directed against conformational epitopes. The mAbs were assayed for their ability to interfere with the binding of PT in model receptor systems like a solid phase binding assay using fetuin as receptor moiety, hemagglutination of chymotrypsin-sensitized goose erythrocytes, and the PT-mediated induction of the clustered growth pattern (CGP) of Chinese hamster ovary (CHO) cells. Five of the eleven mAbs (UB-A1, UB-A2, UB-B7, UB-B12, and UB-D7) interfered with the binding of PT to fetuin on solid phase and with PT-mediated hemagglutination. UB-A2, UB-B7, and UB-B12 also inhibited the induction of the clustered growth pattern of CHO-cells. This indicates that the determinants recognized by these mAbs are associated with the formation of the carbohydrate recognition sites of PT. Thus, the monoclonal antibodies described in this study will be valuable tools in the further analysis of the structure-function relationship of pertussis toxin with respect to receptor recognition and binding.

Animals↗
PubMed1991

Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells.

Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding.

Animals↗
PubMed1991

Identification of linear B-cell determinants of pertussis toxin associated with the receptor recognition site of the S3 subunit.

Receptor recognition of pertussis toxin is mediated by the B oligomer consisting of subunits S2, S3, 2xS4, and S5. One possible way to interfere with toxin action would be the inhibition of recognition and binding of the cellular receptor(s) by preformed toxin-directed antipeptide antibodies. A prerequisite for this approach is the localization of linear antigenic determinants followed by the identification of inhibitory epitopes. Anti-S2 peptide antibodies have been shown to inhibit binding of the holotoxin to in vitro model receptor systems. For the elucidation of linear antigenic and immunogenic determinants harbored in the S3 subunit, synthetic peptides corresponding to selected linear amino acid sequences of S3 have been prepared and used to raise peptide-specific antibodies in rabbits. All peptides elicited a strong homologous response. Four synthetic peptides reacting with anti-pertussis toxin antibodies (R36-51, R87-95, R134-150, and R147-160) have been identified. Seven synthetic peptides (R1-12, R12-23, R14-29m, R36-51, R95-107, R134-150, and R164-178) induced antibodies recognizing pertussis toxin. Thus, these segments correspond to linear antigenic determinants. Analogous to the S2 subunit, the N terminus of S3 proved to be immunorecessive in the native toxin. The highly homologous S2 subunit was only bound strongly in Western blotting (immunoblotting) by antiserum directed at peptide R164-178, which is identical in the S2 and S3 subunits. A weak recognition of S2 in Western blotting was observed with anti-R95-107 antiserum. The ability of affinity-purified anti-S3 peptide antibodies to interfere with pertussis toxin binding was investigated by hemagglutination of goose erythrocytes as a model receptor system for S3-mediated receptor recognition. Antipeptide antibodies directed at R1-12, R12-23, R14-29m, and R36-51 inhibited hemagglutination of goose erythrocytes. This indicates that the corresponding antigenic regions in the S3 subunit are associated with the formation of the receptor binding domain. Inhibition of B-oligomer-mediated pertussis toxin binding to cellular receptors by preformed antipeptide antibodies of sufficient affinity should not only block the detrimental effects of the S1 subunits, but also interfere with the mitogenic effects attributed to the B oligomer.

Amino Acid Sequence↗
PubMed1991

Screening for biotinidase deficiency in children with unexplained neurologic or developmental abnormalities.

To test the hypothesis that the frequency of biotinidase deficiency is greater in children with unexplained developmental delay or neurologic abnormalities than in the general population, we studied children seen at a large outpatient clinic over a four-year period who had one or more of these neurologic abnormalities and for whom no specific cause for their abnormalities could be found. The group totaled 274 children (163 boys; 111 girls) whose ages ranged from 2 weeks to 17 years. Characteristics were IQ/DQ, 30 to 70 in the 115 for whom scores were available; 41% had seizures; 15% had sensorineural hearing loss; 54% showed gross motor delay or ataxia; and 27% had decreased muscle tone. One patient with a classical clinical picture of biotinidase deficiency was diagnosed during the study period and was not included in the study. None of the patients with nonclassic findings had a deficiency of biotinidase activity. Our results suggest that biotinidase deficiency does not account for a large proportion of children with unexplained neurologic abnormalities or developmental delay. This does not negate the importance of biotinidase testing in children with clinical patterns specifically suggestive of the deficiency.

Adolescent↗
PubMed1990

Partial biotinidase deficiency: clinical and biochemical features.

Neonatal screening for profound biotinidase deficiency (less than 10% of the mean normal activity level) has identified a group of children with partial biotinidase deficiency (10% to 30% of mean normal activity). Because partial biotinidase deficiency may result in clinical consequences that may be prevented by treatment with biotin, we evaluated such individuals and their family members (1) to determine whether partial biotinidase deficiency is associated with symptoms and (2) to determine the inheritance pattern. We quantified serum biotinidase activity levels and obtained medical histories of probands, their parents and siblings, and additional family members. All children with partial deficiency were healthy at the time of diagnosis. One child, who was not initially treated with biotin, later developed hypotonia, hair loss, and skin rash, which resolved with biotin therapy. Four adults and three children with partial biotinidase deficiency were identified among family members of infants identified by neonatal screening. All these individuals were healthy, although one sibling had elevated urinary lactate excretion. A fifth adult with partial deficiency, found among clinically normal adult volunteers, later showed minor symptoms that resolved after biotin therapy. Like children with profound biotinidase deficiency, children with partial biotinidase deficiency are symptoms free at birth. However, the subsequent occurrence of symptoms of profound biotinidase deficiency in some persons with partial deficiency suggests that biotin therapy for this condition may be warranted.

Adult↗
PubMed1989

A versatile non-radioactive technique for restriction analysis of recombinant DNA.

Restriction analysis of recombinant DNA is most frequently performed according to Smith and Birnstiel by labeling 5'-termini with 32P, followed by partial digestion, separation, and autoradiographic detection of labeled fragments. We describe a rapid, non-radioactive technique for restriction analysis of recombinant DNA which combines Southern blotting of partial restriction digests and hybridization with a vector-specific probe labeled with the steroid-hapten Digoxigenin for immunological detection. This technique has several advantages compared to conventional methods. Labeling with 32P is not necessary and as the labeled DNA-fragment used as probe is vector-specific, it can be applied for numerous constructs using the particular cloning vector (e.g.pBR322). Furthermore, the probe can be stored for several months and can be reused many times.

Blotting, Southern↗
PubMed1989

Physical mapping of a translocation breakpoint in neurofibromatosis.

The gene for von Recklinghausen neurofibromatosis (NF1), one of the most common autosomal-dominant disorders of humans, was recently mapped to chromosome 17 by linkage analysis. The identification of two NF1 patients with balanced translocations that involved chromosome 17q11.2 suggests that the disease can arise by gross rearrangement of the NF1 locus, and that the NF1 gene might be identified by cloning the region around these translocation breakpoints. To further define the region of these translocations, a series of chromosome 17 Not I-linking clones has been mapped to proximal 17q and studied by pulsed-field gel electrophoresis. One clone, 17L1 (D17S133), clearly identifies the breakpoint in an NF1 patient with a t(1;17) translocation. A 2.3-megabase pulsed-field map of this region was constructed and indicates that the NF1 breakpoint is only 10 to 240 kilobases away from 17L1. This finding prepares the way for the cloning of NF1.

Chromosome Mapping↗
PubMed1989

Autosomal dominant supravalvular aortic stenosis: large three-generation family.

Supravalvular aortic stenosis (SVAS) can be inherited as an isolated autosomal dominant trait or can be a component manifestation of the Williams syndrome. Some consider the Williams syndrome to be due to more severe expression of the gene defect that causes isolated SVAS. We describe a family with isolated SVAS that is the largest thoroughly studied family with this disorder to our knowledge; no patients in this family had Williams syndrome. Five members of this family were reported by Lewis et al. (Dis Chest 55:372-379, 1969). We reevaluated this family and now include examinations of the parents, additional sibs and children of the original 5 patients. Twenty relatives had physical and echocardiographic examinations. In addition, information from outside sources was obtained on 7 relatives not personally evaluated. The SVAS showed marked variability of expression and was not associated with mental retardation or with the facial manifestations of Williams syndrome. We think that previous reports of Williams syndrome reputedly occurring within the same family as isolated autosomal dominant SVAS were inadequately documented. Based on our family and review of the literature, we suggest that isolated SVAS and Williams syndrome represent clinically distinct entities.

Adolescent↗
PubMed1989

Spectrum of findings in a family with nonsyndromic autosomal dominant supravalvular aortic stenosis: a Doppler echocardiographic study.

Nonsyndromic familial supravalvular aortic stenosis is an autosomal dominant disorder. However, for many reported families, systematic study of all family members with echocardiographic or hemodynamic techniques has not been performed and degree of penetrance has not been assessed. The supravalvular stenosis in these family members usually is not associated with mental retardation or other characteristics of Williams syndrome. Although some believe that autosomal dominant supravalvular aortic stenosis is part of the spectrum of Williams syndrome, others believe that these are separate entities. Doppler echocardiograms were analyzed on 23 members of a 34 member family with several known to have supravalvular aortic stenosis; 20 studies were performed by the authors and 3 were done elsewhere and made available for review. No family member had mental retardation, characteristic facies or other findings of Williams syndrome. Three of the 34 had supravalvular aortic stenosis requiring surgery. Of 22 members examined echocardiographically who had not had prior surgical repair, 13 had supravalvular aortic stenosis. Echocardiographic findings ranged widely, from calcification of the ascending aorta in a 71 year old man with minimally increased flow velocity (1.7 m/s) to mild narrowing with mildly increased flow velocity in six members to significant narrowing with impressively increased flow velocity (2 to 4 m/s) in seven. In addition, four patients had mild narrowing of pulmonary artery branches and eight had peak pulmonary artery flow velocity above normal. This study demonstrates complete penetrance with extremely variable expression in this family with autosomal dominant supravalvular aortic stenosis and emphasizes the importance of using echocardiographic techniques in studying the family members who are suspected of having an inherited cardiovascular disease.

Adolescent↗
PubMed1989

Characterization of a translocation within the von Recklinghausen neurofibromatosis region of chromosome 17.

The genetic defect causing von Recklinghausen neurofibromatosis (NF1) has been mapped to the proximal long arm of chromosome 17 by linkage analysis. Flanking markers have been identified, bracketing NF1 in 17q11.2 and laying the foundation for isolating the disease gene. Recently, a family in which a mother and her two children show both the symptoms of NF1 and the presence of a balanced translocation, t(1;17)(p34.3;q11.2), has been identified. We have examined the possibility that the translocation has occurred in or near the NF1 gene by constructing a somatic cell hybrid line containing the derivative chromosome 1 (1qter-p34.3::17q11-qter). On chromosome 1, the breakpoint occurred between SRC2 and D1S57, which are separated by 14 cM. The translocation breakpoint was localized on chromosome 17 between D17S33 and D17S57, markers that also flank NF1 within a region of 4 cM. These data are consistent with the possibility that the translocation event is the cause of NF1 in this pedigree. Consequently, the isolation of the translocation breakpoint, by approach from either the chromosome 1 or the chromosome 17 side, may facilitate the identification of the NF1 gene.

Animals↗
PubMed1989

Inhibition of pertussis toxin binding to model receptors by antipeptide antibodies directed at an antigenic domain of the S2 subunit.

Receptor recognition of pertussis toxin is mediated by the B-oligomer consisting of subunits S2, S3, S4 (two), and S5. To understand the structure-function relationships of the receptor-binding oligomer and to elucidate the immunological structure of pertussis toxin, we assayed antisera generated against each of 10 synthetic peptides corresponding to segments of the pertussis toxin S2 subunit for their ability to recognize the native toxin. Only antisera raised against peptides R1-7, R35-50, and R91-106 recognized pertussis toxin in an enzyme-linked immunosorbent assay and Western blotting (immunoblotting). These segments thus correspond to linear antigenic epitopes. The highly homologous S3 subunit was only weakly recognized by antibodies to R91-106 in Western blotting. The ability of affinity-purified antipeptide antibodies to interfere with the binding of pertussis toxin was investigated by pertussis toxin-mediated hemagglutination of goose erythrocytes, the binding of pertussis toxin to fetuin, and the toxin-induced clustered growth pattern of CHO cells as model receptor systems. Antibodies directed against synthetic peptides R1-7, R35-50, and R91-106 inhibited the binding of pertussis toxin in the two model receptor systems that solely depend on the interactions of the S2 subunit. The toxin-mediated clustered growth pattern of CHO cells could not be inhibited. The results point to a second binding site with distinct specificity involving the S3 subunit of pertussis toxin. The regions identified in this work contribute to the definition of receptor-binding sites of pertussis toxin and should thus improve the development of an acellular-component pertussis vaccine.

Animals↗
PubMed1989

Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin.

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.

Adhesins, Escherichia coli↗