PubMed Health⌕ Search

Biomedical subjects

M A Schmidt

Publications and source records attributed to M A Schmidt.

At least 73 records · Page 4Linked to original sources
PubMed1995

Influence of isometric loading on biceps EMG dynamics as assessed by linear and nonlinear tools.

The summed electrical discharges generated by a contracting skeletal muscle constitute a dynamic system conveying electromyographic (EMG) information indicative of muscle physiological status. "Steady states" of activity can be achieved with light loads, but with heavy loads the dynamic system experiences continuous status transitions that culminate in task failure. The present study was designed to assess the applicability of two mathematical tools, one linear and the other nonlinear, in addressing the time course of EMG alterations under different loading challenges. Surface EMGs of the biceps brachii muscle were recorded from 14 healthy human volunteers during light and heavy loadings, and task failure occurred at varying times among the subjects. Digitized EMG signals were analyzed by linear spectral analysis (fast Fourier transform) and nonlinear recurrence-plot analysis. With light loading, computed variables from both analyses gave "quasi-steady-state" values over time, with recurrence-plot analysis having the higher variance. With heavy loading, the nonlinear variable (%determinism) increased sooner and exhibited larger changes from control values than decreases in the linear variable (spectral center frequency). Experimental results support the conclusion that both analyses can be combined to give a fuller assessment of the biceps EMG during light or heavy loading. Implications for the detection of muscular fatigue are discussed.

Adult↗
PubMed1995

Play ball!

Explore the source record for details and available documents.

Baseball↗
PubMed1994

Elucidation of linear epitopes of pertussis toxin using overlapping synthetic decapeptides: identification of a human B-cell determinant in the S1 subunit indicative of acute infections.

To identify relevant linear epitopes within the immunodominant ADP-ribosyl transferase (S1 subunit) of pertussis toxin (PT), its complete amino acid sequence was synthesized as consecutive, overlapping decapeptides on solid phase and probed for seroreactivity with pertussis specific human antisera in 'peptide scans'. Comparison of the resulting antigenic profiles revealed two distinct types of human antisera, though amino acids 140-200 could not be assessed as the corresponding peptides reacted non-specifically with the detection system. Human anti-pertussis sera predominantly recognized linear immunodominant epitopes located in three separated segments spanning amino acids 3-16, 21-30, and 211-222. Antisera originating from infants with acute B. pertussis infections (type I) identified determinants in all three segments, while type-II antisera from convalescent patients only recognized epitopes in the N-terminal regions. The binding of pertussis specific antisera--both type I and type II--to the holotoxin was inhibited by preincubation of antibodies with synthetic peptides corresponding to two linear determinants located at the N-terminus of S1:R 3-16 and R 21-30. However, competitive binding of antibodies to PT and to synthetic peptides equivalent to the third epitope (R 211-222) was only observed with type I antisera. Thus, the linear immunogenic determinant identified at the C-terminus of the A-protomer represents a human epitope which is apparently specific for antisera from pertussis patients with acute infections. The possible application of this determinant in serologic diagnosis will be a valuable tool to detect and distinguish acute Bordetella pertussis infections.

Adult↗
PubMed1994

Efficient production of active and mutated ADP-ribosyltransferase (S1) of pertussis toxin using affinity expression cassette polymerase chain reaction.

We describe an efficient, general approach for cloning, expression and purification of heterologous proteins in Escherichia coli host strains. The affinity expression cassette polymerase chain reaction (AEC-PCR) allows the insertion of virtually any coding sequence in suitable expression vectors. For ease of purification of the (over)produced protein the gene expression cassettes are engineered by specifically designed oligonucleotide primers in the polymerase chain reaction (PCR) to contain either 3' or 5' additional nucleotides coding for a short amino acid sequence constituting an 'affinity block' fused to either end of the protein. This allows a one-step purification by affinity chromatography. In combination with PCR-mediated site-specific mutagenesis this approach is a highly efficient way for the expression and isolation of proteins and for the analysis and dissection of functional domains. The application of AEC-PCR is demonstrated by the cloning, production and purification of the native, active and the mutagenized, inactive ADP-ribosyltransferase (S1 subunit) of pertussis toxin. In this example, a string of six histidines has been engineered to either the N-terminal or the C-terminal end of the protein to serve as 'affinity block' for the isolation of the recombinant protein by immobilized metal ion affinity chromatography (IMAC). Thus, the S1 subunit can now be produced in sufficient quantities to facilitate further studies on its immunological and molecular properties.

Base Sequence↗
PubMed1993

Diffuse adherence of enteropathogenic Escherichia coli strains--processing of AIDA-I.

The adherence of pathogenic Escherichia coli to the mucosa of the small intestine is an important step in the development of diarrhoea. To study the molecular basis of the diffuse adherence (DA) pattern of E. coli strains expressing the classical serotypes of enteropathogenic E. coli (EPEC), strain 2787 (O126:H27) was investigated. By expression cloning, a plasmid-derived 6.0 kb DNA fragment was identified which conferred the DA phenotype on recipient K-12 strains. This fragment encoded the 100 kDa adhesin involved in diffuse adherence (AIDA-I) which by mild heat shock treatment was isolated from the surface of the wild-type and recombinant DA-positive strains. Analysis of the entire DNA fragment revealed two open reading frames coding for proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as the AIDA-I precursor protein which after cleavage of the signal sequence undergoes additional C-terminal processing for maturation to AIDA-I. Though the function of the cytoplasmic 45 kDa protein is not known, preliminary evidence indicates that authentic expression of the protein is a prerequisite for the correct processing of the 132 kDa precursor to AIDA-I. The AIDA-I precursor exhibits significant homology to the virG (icsA) protein of Shigella flexneri which apparently plays a major role in the events leading to the intercellular spread of invasive Shigella organisms.

Adhesins, Escherichia coli↗
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↗