Biomedical subjects

G F Gerlach

Publications and source records attributed to G F Gerlach.

At least 37 records · Page 2Linked to original sources

PubMed1997

Differentiation of Mycobacterium paratuberculosis isolates by rDNA-spacer analysis and random amplified polymorphic DNA patterns.

The ability to distinguish between isolates of Mycobacterium paratuberculosis isolates was studied using two molecular techniques. Nucleotide sequence analysis of the rDNA-spacer and random amplified polymorphic DNA (RAPD) analysis using decamer primers with GC contents of 60 to 70% were evaluated on 16 isolates of M. paratuberculosis from cattle. The rDNA spacer analysis did not discriminate between isolates as it revealed an identical sequence for all 16 strains tested but it showed one common difference to the sequence previously described for M. paratuberculosis J2A. In the RAPD analysis, 14 of the 60 decamer primers used resulted in distinct amplification products for most of the isolates. For seven of the primers the size of the amplification products varied among strains thus allowing the specific identification of eight of the 16 isolates; of the remaining eight isolates five could each be differentiated from 14 other isolates, two from 13, and one from 12 isolates. Therefore, these data illustrate the possibility of using RAPD-analysis with certain primers for the differentiation of individual M. paratuberculosis isolates.

Animals↗

PubMed1997

Development of an ELISA technique for serodiagnosis of bovine paratuberculosis.

A local clinical Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) isolate was cultivated in large-scale culture. A procedure for efficient preparation of a lipoarabinomannan-containing antigen was developed and standardized; 25 mg of purified antigen were obtained per gram of bacterial wet weight. An ELISA based on this antigen was developed. Intra- and interassay variation were determined to be 20% and 27%, respectively. The ELISA was evaluated using the sera of groups of 39 non-randomly selected and 92 randomly selected animals from which ileocaecal lymph nodes were cultured to isolate viable M. paratuberculosis. Combining the results of both groups a positive predictive value of 74% and a negative predictive value of 99% were calculated. The ELISA was licensed by the German regulatory agencies and, in a direct comparison, was found to be superior to both other licensed assays.

Animals↗

PubMed1997

Characterization of a large transferrin-binding protein from Actinobacillus pleuropneumoniae serotype 7.

The binding of transferrin at the surface of Actinobacillus pleuropneumoniae (A. pp.) is mediated by two proteins of approximately 60 and 100 kDa. The 60 kDa protein has been shown to be highly divergent among different serotypes and to induce a serotype-specific protective immune response. In this study we have characterized the 100 kDa transferrin-binding protein of A. pp. serotype 7 and designated it as TfbB. The tfbB gene was found to be located immediately downstream of the tfbA gene. It was cloned and sequenced, and antibodies raised against the isolated recombinant protein detected, with a constant intensity, a 100 kDa protein in A. pp. serotypes 2, 4, 6, 7, 8, 9, 10 and 11, and a polypeptide of approximately 103 kDa in serotypes 1, 3, 5A and 12. In addition, comparative analysis of the deduced amino acid sequence showed more than 40% identity with the large transferrin-binding proteins of Neisseria meningitidis and Haemophilus influenzae. The TfbB protein was expressed in E. coli outer membranes in a conformation eliciting porcine transferrin-specific binding activity. Sera of pigs immunized with these TfbB-containing E. coli membranes recognized functional membrane-associated TfbB protein whereas no such reaction was observed upon immunization with isolated recombinant TfbB protein. A preliminary animal experiment showed that TfbB-containing outer membrane preparations from recombinant E. coli can reduce significantly the mortality of an A.pp. infection with the homologous strain.

Actinobacillus pleuropneumoniae↗

PubMed1997

Interference of peptides and specific antibodies with the function of the Actinobacillus pleuropneumoniae transferrin-binding protein.

Multiple-antigenic peptides (MAPs) containing transferrin-binding domains of the Actinobacillus pleuropneumoniae serotype 7-derived transferrin-binding protein (TfbA) (K. Strutzberg, L. von Olleschik, B. Franz, C. Pyne, M. A. Schmidt, and G.-F. Gerlach, Infect. Immun. 63:3846-3850, 1995) were constructed. It was found that the MAPs inhibited transferrin binding of the recombinant TfbA protein, whereas antibodies directed against transferrin-binding domains failed to do so.

Actinobacillus pleuropneumoniae↗

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↗

PubMed1995

Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5.

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.

Actinobacillus Infections↗

PubMed1993

Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes.

The three different pore-forming RTX-toxins of Actinobacillus pleuropneumoniae are reviewed, and new and uniform designations for these toxins and their genes are proposed. The designation ApxI (for Actinobacillus pleuropneumoniae RTX-toxin I) is proposed for the RTX-toxin produced by the reference strains for serotypes 1, 5a, 5b, 9, 10 and 11, which was previously named haemolysin I (HlyI) or cytolysin I (ClyI). This protein is strongly haemolytic and shows strong cytotoxic activity towards pig alveolar macrophages and neutrophils; it has an apparent molecular mass in the range 105 to 110 kDa. The genes of the apxI operon will have the designations apxIC, apxIA, apxIB, and apxID for the activator, the structural gene and the two secretion genes respectively. The designation ApxII is proposed for the RTX-toxin which is produced by all serotype reference strains except serotype 10 and which was previously named App, HlyII, ClyII or Cyt. This protein is weakly haemolytic and moderately cytotoxic and has an apparent molecular mass between 103 and 105 kDa. The genes of the apxII operon will have the designations apxIIC for the activator gene and apxIIA for the structural toxin gene. In the apxII operon, no genes for secretion proteins have been found. Secretion of ApxII seems to occur via the products of the secretion genes apxIB and apxID of the apxI operon. The designation ApxIII is proposed for the nonhaemolytic RTX-toxin of the reference strains for serotypes 2, 3, 4, 6 and 8, which was previously named cytolysin III (ClyIII), pleurotoxin (Ptx), or macrophage toxin (Mat).(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus pleuropneumoniae↗

PubMed1993

Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.

Actinobacillus pleuropneumoniae↗

PubMed1992

Immunization of pigs against Actinobacillus pleuropneumoniae with two recombinant protein preparations.

Two Actinobacillus pleuropneumoniae serotype 7 antigens were expressed in Escherichia coli and tested for their protective efficacy in an experimental pig model. The antigens used were a fusion protein containing the carboxy-terminal 70% of the +/- 103 kDa cytolysin and a full length 60 kDa protein which has been shown previously to bind transferrin. Pigs were immunized twice with 25 micrograms of either or both preparations. All pigs developed a strong humoral immune response comparable to that induced by infection. Upon challenge with an A. pleuropneumoniae serotype 7 strain, all immunized groups were less affected by the disease and showed significantly lower mortality than the controls (p less than 0.1). Pigs receiving both antigens had a tendency to recover faster than the controls or animals which were vaccinated with only one antigen. Protection was serotype-specific since no cross-protection was detected following challenge with an A. pleuropneumoniae serotype 1 strain. A dose-response experiment using the single antigens at 200, 50 or 12.5 micrograms per dose showed no difference in protection among the groups.

Actinobacillus pleuropneumoniae↗

PubMed1992

Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4.

Actinobacillus pleuropneumoniae↗

PubMed1992

Characterization of two genes encoding distinct transferrin-binding proteins in different Actinobacillus pleuropneumoniae isolates.

The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli. The nucleotide sequences of the tfbA genes from A. pleuropneumoniae serotypes 7 (G.-F. Gerlach, C. Anderson, A. A. Potter, S. Klashinsky, and P. J. Willson, Infect. Immun. 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity. The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence. The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay. Southern hybridization and Western blot (immunoblot) analysis of the 13 A. pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that of A. pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A. pleuropneumoniae serotype 1. The tfbA genes of A. pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A. pleuropneumoniae serotype 1-derived tfbA probe, and the respective proteins were weakly reactive with the antibody raised against the A. pleuropneumoniae serotype 7 TfbA protein.

Actinobacillus pleuropneumoniae↗

PubMed1992

Gene expression in skeletal muscle in response to stretch and force generation.

Striated muscle is a tissue in which gene expression is influenced to a large extent by mechanical signals. This includes the regulation of gene expression-associated muscle fiber phenotype determination, which depends on which protein isoform genes are transcribed, as well as muscle fiber mass accretion, which appears to involve some translational regulation. Although muscle synthesizes a set of highly specialized proteins it has a remarkable ability to adapt by expressing different isoforms of the same protein so that it acquires the appropriate contractile characteristics. Our work has focused on the myosin heavy chain (HC) genes as these encode the myosin cross bridge, which is responsible for muscle intrinsic velocity of contraction and economy of force development. RNA analyses after cast immobilization of the limb with the muscle in the lengthened or shortened position and/or with electrical stimulation were used to determine the effects of altered mechanical signals on gene transcription. When the soleus muscle was immobilized in the shortened position in the young animal it did not fully differentiate into a slow postural-type muscle. Even in the adult, the soleus muscle if deprived of stretch and contractile activity switches back to transcribing the fast myosin HC gene. The converse was true when the fast rabbit tibialis anterior was subjected to immobilization in the lengthened position and/or electrical stimulation. Both stretch alone and stimulation alone caused repression of the fast type and activation of the slow myosin genes. The reprogramming of the fast muscle was more complete when the stretch was combined with stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Stretch and force generation induce rapid hypertrophy and myosin isoform gene switching in adult skeletal muscle.

Using electrical stimulation to control force generation and limb immobilization to alter the degree of stretch, we have studied the role of mechanical activity in inducing hypertrophy and in determining fast and slow muscle fibre phenotype. Changes in gene expression were detected by analysing the RNA in hybridization studies employing cDNA probes specific for fast and slow myosin heavy chains and other genes. As a result of overload in the stretched position, the fast contracting tibialis anterior muscle in an adult rabbit is induced to synthesize much new protein and to grow by as much as 30% within a period as short as 4 days. This very rapid hypertrophy was found to be associated with an increase of up to 250% in the RNA content of the muscles and an abrupt change in the species of RNA produced. Both stretch alone and electrical stimulation alone caused repression of the fast-type genes and activation of the slow-type genes. it appears that the fast-type IIB genes are the default genes, but that the skeletal slow genes are expressed as a response to overload and stretch. These findings have implications as far as athletic training and rehabilitation are concerned.

Animals↗

PubMed1991

Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7.

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity with a frequency of approximately 10(-4). The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.

Actinobacillus pleuropneumoniae↗

PubMed1991

Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae.

The nucleotide sequence of six genes involved in the expression of type 3 fimbriae of Klebsiella pneumoniae was determined. In addition to the genes that encode the fimbrial subunit (mrkA) and adhesion (mrkD), the mrkB, mrkC, and mrkE genes appear to be involved in assembly of the fimbrial filament and regulation of type 3 fimbrial expression. The mrkF gene product is required to maintain the stability of the fimbrial filament on the cell surface.

Adhesins, Bacterial↗

PubMed1990

Mechanisms of temperature acclimation in the carp: a molecular biology approach.

Most fish are unable to regulate their body temperature. Therefore their muscle power output would be expected to decline markedly with decreased environmental temperature. However, some species including carp show a remarkable ability to acclimate. The nature and significance of changes in enzymatic myosin ATPase activity and the recruitment of different muscle fiber types in relation to acclimation and environmental temperature in carp are reviewed. These changes presumably involve qualitative and quantitative changes in gene expression. To approach the problem, we have constructed a carp genomic library and isolated 28 nonoverlapping clones containing sequences of different myosin heavy chain (MHC) isoforms along with a clone containing carp actin sequences. Plasmids containing probes specific for a carp fast muscle MHC and actin RNA were constructed. Hybridization results suggest that the expression of this MHC isoform RNA is increased in warm- relative to cold-acclimated carp. These data indicate that there are different MHC isoform genes that are expressed at warm and cold environmental temperatures.

Acclimatization↗

PubMed1989

Type 3 fimbriae among enterobacteria and the ability of spermidine to inhibit MR/K hemagglutination.

The distribution of the gene cluster encoding type 3 fimbriae among various isolates of the family Enterobacteriaceae was investigated by using 112 clinical and nonclinical isolates. Closely related DNA sequences were detected in all Klebsiella strains, in most Enterobacter isolates, in a smaller number of Escherichia coli and Salmonella spp., and in a single isolate each of Yersinia enterocolitica and Serratia liquefaciens but not in isolates of Morganella or Providencia species or Serratia marcescens. Except for E. coli and Salmonella strains, the presence of gene sequences was correlated with the phenotypic expression of either the MR/K hemagglutinin or fimbriae that reacted with specific antibodies. In one isolate of Y. enterocolitica the expression of type 3 fimbriae was plasmid determined. The polyamine spermidine was identified as an inhibitor of MR/K hemagglutinating activity, exhibiting an MIC of 1.2 mM. Spermidine inhibited the hemagglutination of 37 MR/K-positive clinical isolates from various genera. However, one clinical isolate of Enterobacter cloacae and most (four of five) nonclinical Klebsiella isolates were not completely inhibited.

Antibodies, Monoclonal↗

PubMed1989

Expression of type 1 fimbriae and mannose-sensitive hemagglutinin by recombinant plasmids.

Deletions within the cloned genes (fimA) encoding the type 1 major fimbrial subunits of two isolates of Klebsiella pneumoniae resulted in a nonfimbriate but hemagglutinating phenotype after transformation of Escherichia coli HB101 or ORN103. Phenotypic expression of type 1 fimbriae could be restored by transformation with plasmids containing the fimA genes of the fimbrial gene clusters from different strains. The surface fimbriae expressed were serologically identical to those of the polymerized product of the introduced fimA gene. The fimA gene products of Salmonella typhimurium and Serratia marcescens could utilize the accessory fimbrial genes of K. pneumoniae to produce surface-associated, hemagglutinating fimbriae. The relatedness of the type 1 fimbrial gene clusters from multiple isolates of members of the family Enterobacteriaceae was examined by DNA hybridization techniques. These analyses demonstrated little nucleotide sequence agreement among distinct genera of the enteric bacteria.

Bacterial Adhesion↗