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J Frey

Publications and source records attributed to J Frey.

At least 181 records · Page 10Linked to original sources
PubMed1993

Receptor binding of osteoblast-specific factor 1 (OSF-1/HB-GAM) to human osteosarcoma cells promotes cell attachment.

OSF-1/HB-GAM is a member of developmentally regulated growth factors and cytokines. High expression levels of this factor are found in different tissues, e.g., in brain and in bone. We have analyzed the biological function and binding properties of natural OSF-1 to human osteoblasts. Using antibodies raised against the entire OSF-1 molecule or a synthetic carboxy-terminal peptide (amino acids (aa) 110-140) we have investigated the binding sites of rat OSF-1 on human osteoblast-like osteosarcoma cell lines: HOS(TE85) and MG-63. Immunofluorescence microscopic studies and flow cytometric data revealed that OSF-1 is specifically bound to the surface of these cells. Further characterization of the binding sites showed that both osteosarcoma cells express two different kinds of binding sites: Besides binding to a specific OSF-1 receptor, OSF-1 also significantly binds to cell surface heparan sulfates. Using the peptide specific polyclonal antibody we show that the carboxy-terminal domain, aa 110 to 140 of OSF-1, seems to be involved in ligand binding. Studies on the biological function of OSF-1 revealed a strong cell attachment promoting activity in vitro. This activity is not diminished after digestion of cell surface heparan sulfates by heparinase I and heparitinase I, demonstrating that the OSF-1 receptor mediates the cell attachment of osteoblasts. Our results indicate that one biological function of OSF-1 is the promotion of osteoblast attachment to the extracellular bone matrix.

Amino Acid Sequence↗
PubMed1993

[Evaluation of urinary calcium/creatinine ratio in premature and full-term newborn infants].

Urinary calcium/creatinine ratio (U Ca/creat) was studied in infants, term and preterm newborn babies. In 31 1.5-24 months old healthy infants, the median U Ca/creat was 0.16 mmol/mmol (range 0.013-1.17) and was similar to the value obtained in children older than 4 years. In 55 healthy full-term newborns studied in the first week of life, the median U Ca/creat was 0.12 mmol/mmol. However the range of values was extremely wide (0.0006-4.75), suggesting that the U Ca/creat ratio is of little interest to screen for hypercalciuria during the neonatal period. In 31 premature newborns, the median U Ca/creat was 1.08 mmol/mmol, a value significantly higher than in the two other groups (P < 0.001); as in the term newborns there was a very wide range of values (0.057-6.83). However after excluding the premature babies with elevated serum 25 OHD level, this difference was not statistically significant.

Age Factors↗
PubMed1992

[The place of percutaneous transluminal angioplasty in the treatment of obliterative vascular diseases of the lower limbs. Angioplasty of of the iliac system].

Between 1983-91 386 patients with hemodinamically significant stenosis or short-segment occlusion of iliac arteries were treated by transluminal angioplasty. 487 dilatations and 13 recanalizations were performed. In 353 (70.2%) of these cases were carried out without medicinal preparation continuously after the diagnostic angiography. The technical success rate was 93.4%. The technical success rate did not depend on medicinal preparation. 93% of their patients became symptomless or improved. 28 complications were noted (7 thrombosis, 4 embolisms, 14 inguinal haematomas, 2 false aneurysms, 1 bleeding), 11 of them required surgical intervention. One patient died because of cardiac failure due to retroperitoneal haematoma. Authors' opinion isolated short uni- and bilateral iliacal stenosis can be treated by transluminal angioplasty. Longer or multifocal stenosis required individual consideration.

Aneurysm↗
PubMed1992

Replication and copy number control of the broad-host-range plasmid RSF1010.

Initiation of replication of the broad-host-range plasmid RSF1010, is accurately controlled by the plasmid-encoded proteins, RepB (MobA), RepB', RepA and RepC [Haring et al., Proc. Natl. Acad. Sci. USA 82 (1985) 6090-6094; Scherzinger et al., Nucleic Acids Res. 19 (1991) 1203-1211]. The genes encoding these proteins which are essential for replication and conjugative mobilization are transcribed from a cluster of promoters, P1/P3 and P2, which partly overlap with the origin of conjugal transfer, oriT. Three regions were found where deletion mutations affect the mobilization of RSF1010 and increase its copy number in Escherichia coli. A deletion in the mobC gene increased the copy number of RSF1010 four-fold. Another deletion, that removed oriT and part of the promoter believed to be responsible for the expression of mobC, results in a three-fold increase in copy number. The third type of deletions affect the N-terminal part of RepB (MobA). A deletion that created a frame-shift results in a three-fold increase in copy number. A smaller, in-frame deletion of this region only affected the mobilization of RSF1010, but not its copy number. The extent by which RSF1010 or its deletion derivatives could repress the P1/P3 and P2 promoters has indicated that these promoters are negatively regulated by MobC and RepB (MobA), presumably by their attachment to the oriT region of RSF1010. Both MobC and RepB are required for the maximal repression of the rep operon. Optimal function of RSF1010 thus involves not only overlapping genes, but also proteins that exert multiple functions, mobilization, replication and regulation.

Amino Acid Sequence↗
PubMed1992

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.

Animals↗
PubMed1992

Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.

Actinobacillus pleuropneumoniae↗
PubMed1992

Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1.

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher in A. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant.

Actinobacillus pleuropneumoniae↗
PubMed1992

[Synthesis and supramolecular organization of components of the extracellular matrix by fibroblasts cultured in a collagen lattice].

Fibroblasts cultured in a collagen gel contract and organize the gel into a three-dimensional matrix of collagen fibers. Within this matrix, the fibroblast cell cycle is blocked at the G1 phase but also at the G2 phase. The fibroblasts produce the main extracellular matrix components (collagen, noncollagen proteins, glycosaminoglycans), although in small amounts. Studies using this in vitro model with radiolabeled precursor substances (14C proline, 3H glucosamine) demonstrated production of supermolecular complexes which resisted to proteolysis by pepsin and collagenase and could not be isolated by saline precipitation. Polyclonal antibodies identified type I collagen, type VI collagen and fibronectin in this coherent supermolecular structure. The presence of glycosaminoglycans was also demonstrated by alcian blue precipitation.

Cells, Cultured↗
PubMed1991

Identification and partial characterization of the hemolysin (HlyII) of Actinobacillus pleuropneumoniae serotype 2.

The secreted hemolytic activity produced by Actinobacillus pleuropneumoniae serotype 2 reference strain is thermolabile, inactivated by proteinase K and requires Ca2+ as cofactor for its hemolytic activity. Purification of the hemolytic activity resulted in a fraction containing two proteins, one of 105 kDa and one of 125 kDa. These two proteins could be further separated by preparative SDS polyacrylamide gel electrophoresis. This purification step, resulted in loss of the hemolytic activity. Polyclonal antibodies were made against each of these proteins in rabbits. Neutralization experiments showed that antibodies made against the 105 kDa protein could neutralize the hemolytic activity produced by A. pleuropneumoniae serotype 2, while antibodies made against the 125 kDa protein were unable to neutralize the hemolytic activity. The 105 kDa protein therefore, is the hemolysin of A. pleuropneumoniae serotype 2, known as HlyII. This protein is closely related immunologically to the hemolysin I (HlyI) from A. pleuropneumoniae serotype 1. DNA::DNA hybridization experiments performed by the Southern blot method using the cloned structural gene of HlyI from A. pleuropneumoniae serotype 1 demonstrate that the structural genes of the two hemolysins (hlyIA and hlyIIA) are different and show at least 30% heterology. This confirms that HlyI and HlyII are two different proteins, although they have a very similar molecular weight and show strong immunological cross reactions.

Actinobacillus↗
PubMed1991

Molecular basis of IgG Fc receptor III defect in a patient with systemic lupus erythematosus.

By flow cytometry analysis we could show a decreased expression of Fc gamma receptor type III (Fc gamma RIII) on granulocytes of a patient with systemic lupus erythematosus (SLE). Therefore, we constructed a leukocyte cDNA library from this patient with the aim of investigating this defect on the molecular level. Using an Fc gamma RIII cDNA probe we isolated 15 Fc gamma RIII cDNA clones, which were all characterized by sequencing. Our sequence data revealed that the patient was heterozygous for Fc gamma RIII (NA-1/NA-2). Only clone 5 (NA-2) was a full-length cDNA clone. In contrast to the wild-type Fc gamma RIII the signal sequence is mutated, lacking the hydrophobic region essential for co-translational transport across the endoplasmic reticulum membrane. The predicted transport defect leading to the lack of membrane expression could be confirmed by immunofluorescence staining after expression of this cDNA clone in BHK cells. The cDNA clones 6 and 8 (NA-1) lack the first 45 bp of the signal sequence, but considering the flow cytometry data the signal sequence must be functional allowing the membrane expression of this receptor allele. The part of the cDNA sequence of all isolated clones coding the mature Fc gamma RIII is identical to the wild-type sequence. Therefore, we conclude that the decreased expression of Fc gamma RIII on granulocytes of this SLE patient is due to the transport defect of one of the receptor alleles.

Amino Acid Sequence↗
PubMed1991

Structural requirements of the cytoplasmic domains of the human macrophage Fc gamma receptor IIa and B cell Fc gamma receptor IIb2 for the endocytosis of immune complexes.

Two isotypes of the monocyte/macrophage as well as B cell Fc gamma receptor type II (FcRIIa and FcRIIb2, respectively) mainly differ in the length (76 vs. 44 amino acids) and amino acid sequence of their cytoplasmic domains. Only the eight amino acids just behind the putative transmembrane region are identical. Despite this marked difference, both FcRII mediate endocytosis of immune complexes. To determine the functional significance of the cytoplasmic domains, we expressed truncated FcRIIa and FcRIIb2 in FcR- BHK-21 cells. Mutants of both receptors containing only one amino acid (tail-minus) of the cytoplasmic domain failed to mediate immune complex uptake. The significance of the cytoplasmic domain of the receptors could be further demonstrated using a chimeric FcRIII-FcRIIa construct. Therefore we expressed an FcRIII lacking the hydrophobic carboxyl terminus (containing the putative phosphatidyl - inositol - glycan anchor site) fused inframe to the transmembrane and cytoplasmic domain of the FcRIIa in BHK-21 cells. In contrast to the wild type FcRIII, this chimeric receptor mediated immune complex uptake indistinguishable from that mediated by the FcRIIa. Receptor mutants with relatively short cytoplasmic domains (FcRIIb2: 13, and FcRIIa: 16 amino acids) revealed, that these short amino acid stretches are sufficient to allow reduced receptor-mediated endocytosis of bound ligand. Furthermore, using FcRIIa deletion mutants with a cytoplasmic domain consisting of 62, 46, and 28 amino acids, respectively, we found that the capability of these mutants to mediate immune complex uptake decreased gradually with the truncation of the cytoplasmic tails. Thus, only short amino acid sequences of the cytoplasmic domain are sufficient to enable an, albeit reduced, receptor-mediated endocytosis.

Animals↗
PubMed1991

Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.

The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.

Actinobacillus↗
PubMed1991

Factors involved in immunity against Actinobacillus pleuropneumoniae in mice.

Active and passive immunization studies in mice were undertaken to examine the protective efficiency of vaccines prepared from different components of Actinobacillus pleuropneumoniae, or combinations thereof. Subcutaneous immunization using either washed formalinized whole cells, capsular polysaccharide, lipopolysaccharide or purified hemolysin I (105 kDa protein) partially protected mice against intranasal challenge with a lethal dose of homologous or heterologous A. pleuropneumoniae serotypes. However, full protection was obtained if the formalinized whole cells were supplemented with purified hemolysin. Similar protection was obtained when mice were immunized simultaneously with a sublethal dose of live cells by the intranasal route and with formalinized whole cells subcutaneously. Passive immunization using rabbit hyperimmune serum against formalinized whole cells provided almost total protection whereas hyperimmune serum against capsular polysaccharide, lipopolysaccharide or hemolysin alone provided only a partial protection. Cell mediated immunity as detected by the foot pad test may not be implicated significantly in the protein against acute A. pleuropneumoniae infection. However, humoral immune response seems to play an important role in protection. All the antigenic components examined may contribute to the protection to some extent. However, heat-labile components such as hemolysin and outer membrane proteins may play a crucial role in protection against acute challenge infection.

Actinobacillus Infections↗