Biomedical subjects

J Frey

Publications and source records attributed to J Frey.

At least 145 records · Page 8Linked to original sources

PubMed1995

Effects of gamma-alumina and hydroxyapatite coatings on the growth and metabolism of human osteoblasts.

The behavior of human osteoblasts cultivated on hydroxyapatite or alumina-coated disks of Ti6AL4V was studied in vitro. Cell anchorage and spreading were observed by scanning electron microscopy. Cell growth was monitored by counting cells and measuring DNA at 5 h and 2,5, and 10 days after cell seeding. Cells grown for 10 days were labeled with 14C-proline and total protein and collagen synthesis were measured; the type of collagen was also determined. Both ceramics showed excellent biocompatibility. At 10 days of culture the cells showed a higher rate of proliferation on alumina than on hydroxyapatite. Neither ceramic altered the collagen biosynthesis or the osteoblast-like properties of the cells, as indicated by the high percentage of type I collagen.

Aluminum Oxide↗

PubMed1995

Collagen biosynthesis in cell culture from chorionic villi.

After chorionic villus biopsy of human placenta, cell cultures were propagated with Ham's F10 medium or Eagle's minimum essential medium (MEM). It was possible to study the morphology of the cells by transmission electron microscopy (TEM) after a special culture of the cells in a collagen gel. The cells embedded in a collagen gel were able to contract the gel and to organize collagen fibres, as fibroblast cultures do. TEM showed vacuolization and well-developed cisternae of the endoplasmic reticulum, especially in the case of MEM culture. The aim was to determine whether cells cultivated from early placenta were able to synthesize enough collagen for a metabolic study. A high level of collagen biosynthesis could be quantified. Types I and III collagen can be determined which is useful for studying the abnormalities of collagen synthesis in suspected cases of osteogenesis imperfecta or Ehlers-Danlos type IV syndrome. The hydroxylation of lysine can also be studied with respect to Ehlers-Danlos type VI syndrome. Moreover, these cells, in contrast to fibroblast cultures, made it possible to study the biosynthesis of type IV collagen.

Cells, Cultured↗

PubMed1995

Collagen bioassay by the contraction of fibroblast-populated collagen lattices.

The ability of fibroblasts to induce contraction of a collagen gel was studied with respect to the quantity and the quality of type I acid-soluble collagen. The speed of contraction and the appearance of the fibre bundles obtained after contraction depend not only on the ratio of the amount of collagen to fibroblasts but also on the process of the collagen purification. When collagen lattices made with a pepsinized collagen were compared to lattices made with a non-pepsinized collagen of the same amount, the fibres from pepsinized collagen seemed fewer (only 13% of the observed surface against 51% in the case of non-pepsinized collagen) and the lattices appeared by electron microscopy to be almost empty as if the lattices were comprised of less collagen. The importance of the non-helical domain of the collagen molecule for the identification and organization of collagen by fibroblasts is discussed. Collagen retained by fibroblasts was maximum (80-99%) when collagen was prepared in the presence of protease inhibitors and decreased when proteolysis was not avoided, for example when collagen was prepared in the presence of pepsin. A test using an estimation of the percentage of collagen retained by fibroblasts in a contracted collagen lattice is proposed to check the biological quality of collagen samples.

Acetates↗

PubMed1995

Sodium channels and GABAA receptor-channel complex as targets of environmental toxicants.

Voltage-activated sodium channels and GABAA receptor-chloride channel complex are among the most important target sites of various environmental neurotoxicants. Pyrethroids keep the sodium channels open for prolonged periods of time leading to hyperexcitation of the entire nervous system. In rat cerebellar Purkinje neurons and dorsal root ganglion neurons, only about 1% of sodium channel population needed to be modified by the pyrethroid tetramethrin to increase the depolarizing after-potential to the level of the threshold membrane potential for generation of repetitive after-discharges. This concept of toxicity amplification is applicable to other chemicals that go through a threshold phenomenon to exert their effects. The potency of pyrethroids on neuronal sodium channels increased with lowering the temperature with a Q10 value of 0.2. The selective pyrethroid toxicity between mammals and insects can be quantitatively explained on the basis of the differences in 5 factors, i.e. the intrinsic sodium channel sensitivity, the sodium channel modification due to temperature difference, the reversibility of sodium channel, the detoxication of pyrethroids, and body size. These 5 factors are multiplied to approximately 2000 which is in the same order of magnitude as that of the difference in LD50. Dieldrin had a dual action on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The initial transient potentiation of GABA-induced currents after application of dieldrin was followed by a suppression. Dieldrin-induced potentiation of current was observed only when the gamma 2 subunit was present in embryonic kidney cells (HEK-293) transfected with GABA receptor subunits. Dieldrin-induced suppression was observed in the presence and absence of the gamma 2 subunit. The dieldrin suppression of GABA-induced currents is deemed directly responsible for hyperactive symptoms of poisoning in animals.

Animals↗

PubMed1995

Molecular investigation of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniae serotype 5.

The extracellular hemolytic toxins (ApxI and ApxII) of Actinobacillus pleuropneumoniae are thought to be important factors in this microorganism's virulence and the pathogenesis of swine pleuropneumonia. Using the polymerase chain reaction, the apxI locus of a non-hemolytic, avirulent mutant of A. pleuropneumoniae serotype 5 (mIT4-H) generated by chemical mutagenesis (Inzana T. J., Todd J., Veit H. P. Microb Pathog 1991; 10: 281-96) was found to contain deletions that affected major parts of the entire apxICABD operon, thus inactivating each gene in the operon. The apxII locus was not affected. Monoclonal antibodies to ApxI and ApxII were used to confirm that ApxI was not synthesized, and that ApxII was synthesized but not secreted from the cell. The apxICABD genes and apxIBD genes were cloned into a broad host range vector to obtain plasmids pJFF800 and pJFF801, respectively. Each recombinant plasmid was electroporated into strain mIT4-H to obtain strain mIT4-H/pJFF800 and strain mIT4-H/pJFF801, respectively. Strain mIT4-H/pJFF800 exported ApxI and ApxII, and produced hemolytic activity comparable to or exceeding that of wild type strain J45. Strain mIT4-H/pJFF801 exported only ApxII and produced weak hemolytic activity. Strain mIT4-H/pJFF800 was virulent in mice, and had an LD50 of about 2 x 10(6) colony forming units. In contrast, mIT4-H/pJFF801 and mIT4-H were essentially avirulent in mice, and LD50s for these strains could not be calculated. Strain mIT4-H/pJFF800 was virulent in pigs and caused lethal pleuropneumonia, whereas parent strain mIT4-H was avirulent. Strain mIT4-H/pJFF801 was also able to induce pleuropneumonia in pigs, although a higher dose was required to induce lesions similar to those caused by mIT4-H/pJFF800. Thus, A. pleuropneumoniae strains that produce ApxI and ApxII require ApxI for full virulence and toxic activity in pigs. However, other factors including ApxII contribute to the virulence of A. pleuropneumoniae in pigs.

Actinobacillus Infections↗

PubMed1995

Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains.

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

Actinobacillus pleuropneumoniae↗

PubMed1995

Virulence in Actinobacillus pleuropneumoniae and RTX toxins.

RTX toxins are pore-forming, cytolytic protein toxins that occur widely among pathogenic Gram-negative bacteria. RTX toxins appear to play a direct role in the virulence of Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia. This discovery has led to the development of new diagnostic and epidemiological tools, as well as vaccines, that are useful for a broad variety of serotypes.

Actinobacillus pleuropneumoniae↗

PubMed1995

Restriction fragment length polymorphisms among the flagellar genes of the Lior heat-labile serogroup reference strains and field strains of Campylobacter jejuni and C. coli.

Several typing systems have been described for Campylobacter jejuni and C. coli, to assess the complex epidemiology of these important enteric pathogens. In the present study two typing methods, slide agglutination according to the Lior scheme, and the demonstration of restriction-fragment length polymorphisms (RFLP) of flagellar genes, have been used in parallel on a set of 194 strains. This set comprised 118 sero-reference strains of C. jejuni and C. coli of the Lior scheme, as well as 76 clinical isolates. All isolates were serotyped and subjected to PCR for amplification of flagellar genes, and the PCR product was restricted with Alu I. Flagellar genes could be amplified in 152 strains. Among 85 seroreference strains, 74 different RFLP patterns were observed, and among 67 clinical isolates, there were 36 patterns. There was only limited correlation between flagellar RFLP and the Lior serogroup, and the variability of patterns in serogroups HL2 and HL4 were as marked as the variability between serogroups. Flagellar gene RFLP patterns are shown to be stable, highly discriminatory epidemiologic markers.

Animals↗

PubMed1995

Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment.

The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus-host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle.

Adenoviridae Infections↗

PubMed1995

Insertion element IS1296 in Mycoplasma mycoides subsp. mycoides small colony identifies a European clonal line distinct from African and Australian strains.

Strains of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the agent of contagious bovine pleuropneumonia (CBPP), were analysed with respect to the polymorphism of distribution of a newly discovered insertion element, IS1296, on the chromosome. Analysis of 64 strains isolated from Europe, Africa and Australia, including four vaccine strains and the type strain PG1, revealed ten different IS patterns, forming two main clusters. The European strains originated from outbreaks of CBPP in different countries, and from various other sources such as semen and preputial washings from cattle, lungs from goats and buffalo, and milk from sheep. They showed identical IS1296 patterns, except one strain which had an additional IS1296 element, but the pattern belonged to the same cluster. This shows that the strains from Europe form a clonal lineage. The strains originating from different geographical parts of the African continent and from Australia showed four closely related IS1296 patterns which belong to a separate cluster. This indicates that strains from Africa and Australia form a clonal lineage different from that of the European strains, suggesting that the sporadic cases of CBPP that have re-emerged in Europe almost 15 years after the last declared endemic case in 1967 arose from an established reservoir within Europe rather than being the result of repeated importation from Africa and Australia. While most strains from Africa and Australia had the same IS1296 pattern, all vaccine strains could be distinguished by an individual pattern. The type strain PG1 also had a particular IS1296 pattern which belongs to the cluster of the strains from Africa and Australia. The molecular definition of clonality of M. mycoides subsp. mycoides SC strains with IS1296 represents a rapid and reproducible method for subtyping and differentiation of vaccine strains. It permits at the present time the definition of two main clonal lines, one including the strains from the European continent and a second with strains from Africa and Australia.

Africa↗

PubMed1995

Features of the DNA fingerprinting probe pITZ1.

Stringently controlled plasmids generate DNA fingerprint patterns in mammals when used at low hybridization temperatures. In order to develop a probe for use in paternity testing in cattle we screened a bovine, partial genomic plasmid library with the PCR-amplified ori region of plasmid P1. Of eight isolated clones one generated strong band patterns at high stringency in various mammalian species (data not shown). Sequence analysis revealed an imperfect, compound dinucleotide repeat region, which was PCR-amplified and cloned into the plasmid vector pUC19. Fingerprint results generated by this probe (termed pITZ1) in cattle are compared with the results generated by VNTR-probe pV47, which itself was developed by screening a human chromosome 16 library with tandem repeats of bacteriophage M13. Probe pITZ1 is useful in conjunction with other VNTR-probes for DNA fingerprinting in cattle and donkey populations. The dinucleotide repeat region responsible for the band patterns generated with pITZ1 is close to an Alu-like sequence, which may be involved in eukaryotic replication mechanisms.

Animals↗

PubMed1995

Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR).

Clostridium perfringens type C, which produces alpha- and beta-toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans. A polymerase-chain-reaction (PCR) assay was developed for the specific detection of the genes encoding alpha-, beta-, epsilon- and entertoxin of C. perfringens for rapid typing of C. perfringens strains, and especially for the identification of type C strains. Both the alpha- and beta-toxin genes were detected directly in porcine C. perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 10(3) cells without purification of the DNA. The alpha-toxin gene was detected in all types of C. perfringens. The epsilon-toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains. Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay. The alpha- and beta-toxin genes PCR assay were used to identify C. perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea. At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C. perfringens type C infection. All of them had C. perfringens which gave a positive PCR signal for alpha- and beta-toxin genes, and, hence, were identified as type C strains. From the 16 other piglets with lesions other than necrotizing enteritis, C. perfringens strains with the alpha-toxin gene, but no beta-toxin gene, were isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Rapid and accurate identification of Escherichia coli K-12 strains.

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.

Animals↗

PubMed1995

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.

Adaptation, Physiological↗

PubMed1995

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni.

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Amino Acid Sequence↗

PubMed1995

Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing the 24-kDa protein reacted with C. jejuni, whereas C. coli did not react specifically. Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed an open reading frame encoding 214 amino acids; the gene was designated mapA; and its gene product was designated MAPA. The 18 N-terminal amino acid residues constitute a signal sequence characteristic of prokaryotic membrane lipoproteins. In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni were unequivocally discriminated from 126 other campylobacters, including 34 C. coli isolates. A PCR test based on the mapA sequence was developed for identification of C. jejuni. A PCR product was obtained with all of the clinical isolates of C. jejuni tested from human, dog, cat, bovine calf, and chicken sources. Recombinant MAPA with an added C-terminal six-histidine tail was affinity purified and used to immunize rabbits. The rabbit anti-MAPA serum specifically recognized the protein in whole cells of C. jejuni on Western blots (immunoblots). The MAPA protein was present in all of the C. jejuni strains tested and was absent in C. coli and related campylobacters.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of the human gene encoding aspartyl beta-hydroxylase.

Sequence information for aspartyl beta-hydroxylase (AspH), which specifically hydroxylates one Asp or Asn residue in certain epidermal growth factor (EGF)-like domains of a number of proteins, is so far only described for bovine species. We have isolated a 4.3-kb cDNA encoding the human AspH (hAspH) by immunoscreening of a human osteosarcoma (MG63) cDNA library in lambda ZAP with an antiserum raised against membrane fractions of these cells. Northern blot analyses revealed two transcripts with lengths of 2.6 and 4.3 kb. The deduced amino acid (aa) sequence of this cDNA encodes a protein of 757 aa (85 kDa). Comparison with the deduced bovine AspH (bAspH) aa sequence showed striking differences in the N-terminal portion of this protein. In vitro transcription and translation in the presence of canine pancreas microsomes yielded a 56-kDa protein. Western blot analyses of membrane fractions from MG63 cells with AspH-specific antibodies revealed a protein of the same M(r). These results suggest a posttranslational cleavage of the catalytic C terminus in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1994

Tyrosine-containing sequence motifs of the human immunoglobulin G receptors FcRIIb1 and FcRIIb2 essential for endocytosis and regulation of calcium flux in B cells.

Human B cells express two closely related immunoglobulin G receptors, FcRIIb1 and FcRIIb2, which differ by a 19 amino acid insertion in the cytoplasmic tail of FcRIIb1. The cytoplasmic tails of both isoforms contain a conserved sequence motif (AENTITYSLL) essential for mediating endocytosis via FcRIIb2. Truncation of this motif abolished endocytosis, while replacement of tyrosine (Tyr273) in FcRIIb2 by phenylalanine had no effect on the amount and kinetics of ligand uptake. Co-cross-linking of FcRIIb1 or FcRIIb2 with the antigen receptor on B cells led to an abortive calcium signal. Neither isoform interfered with the early intracellular calcium mobilization, but both prevented the opening of a plasma membrane calcium channel essential for a sustained elevated intracellular calcium level. Modulation of calcium channel activity is mediated by the same sequence motif essential for endocytosis but requires the presence of Tyr292 in FcRIIb1 and Tyr273 in FcRIIb2. Co-cross-linking of FcRIIb1 with surface IgG is associated with tyrosine phosphorylation of Tyr292, whereas Tyr272 in FcRIIb2 was not phosphorylated. Thus, FcRIIb phosphorylation is probably not directly involved in the modulation of the calcium signal but may be essential for further diversification of signals transduced via the coexpressed isoforms FcRIIb1 and FcRIIb2.

Animals↗