Biomedical subjects

J Frey

Publications and source records attributed to J Frey.

At least 55 records · Page 3Linked to original sources

PubMed2001

Work interest as a predictor of competitive employment: policy implications for psychiatric rehabilitation.

Consumers with serious mental illness (N = 166) enrolling in two community-based mental health programs, a vocational Program of Assertive Community Treatment and a clubhouse certified by the International Center for Clubhouse Development (ICCD), were asked about their interest in work. About one third of the new enrollees expressed no interest in working. Equivalent supported employment services were then offered to all participants in each program. Stated interest in work and receipt of vocational services were statistically significant predictors of whether a person would work and how long it would take to get a job. Two thirds of those interested in work and half of those with no initial interest obtained a competitive job if they received at least one hour of vocational service. Once employed, these two groups held comparable jobs for the same length of time. These findings demonstrate the importance of making vocational services continuously available to all people with serious mental illness, and the viability of integrating these services into routine mental health care.

Adult↗

PubMed2001

Clinical responsibility and client autonomy: dilemmas in mental health work at the margins.

Mental health outreach to homeless persons requires practice standards for cases in which clinical assessment and client autonomy conflict. After reviewing the principles of mental health outreach and presenting case examples, conditions and boundaries within which outreach workers negotiate the clinical responsibility/client autonomy dilemma are discussed. Guidelines to support sound clinical practice while respecting client autonomy are also discussed.

Community-Institutional Relations↗

PubMed2001

The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro.

The inositol polyphosphate 5-phosphatase SHIP plays an important role in negative signalling in B cells and mast cells and in the down-regulation of cytokine receptor-mediated signals in myeloid cells. SHIP is expressed as a 145 kDa full-length protein and an isoform of 135 kDa due to alternative splicing. Additional smaller forms of SHIP which are truncated at the carboxy terminus have been described in bone marrow and peripheral blood mononuclear cells (PBMC). Our data demonstrate that human bone marrow cells and PBMC from healthy donors and patients with acute myeloid leukemia express the 145 kDa form of SHIP and low amounts of a 135 kDa form of SHIP in vivo whereas C-terminal-truncated SHIP proteins are generated by a PMSF-sensitive protease during the preparation of cell lysates in vitro. We have further characterized this protease and identified a proteolytic cleavage site in the human SHIP protein C-terminal to tryptophan residue 941. These data support a physiological role for the 145 and 135 kDa forms of SHIP in bone marrow and peripheral blood cells from normal donors and patients with acute myeloid leukemia.

Alternative Splicing↗

PubMed2001

C-terminal region of Pseudomonas aeruginosa outer membrane porin OprD modulates susceptibility to meropenem.

We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 microg/ml) but susceptible to meropenem (<1 microg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid L-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.

Amino Acid Sequence↗

PubMed2001

Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence.

Highly virulent strains of Mycoplasma mycoides subsp. mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-(14)C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake. L-alpha-Glycerophosphate was not transported by M. mycoides subsp. mycoides SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H(2)O(2). Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H(2)O(2) was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains. M. mycoides subsp. mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.

ATP-Binding Cassette Transporters↗

PubMed2001

Antimicrobial growth promoter ban and resistance to macrolides and vancomycin in enterococci from pigs.

Ninety-six enterococcus isolates from fecal samples of pigs receiving tylosin as an antimicrobial growth promoter and 59 isolates obtained in the same farms 5 to 6 months after the ban of antimicrobial growth promoters in Switzerland were tested for susceptibility to nine antimicrobial agents. A clear decrease in resistance to macrolides, lincosamides, and tetracycline was visible after the ban. Vancomycin-resistant Enterococcus faecium belonged to the same clonal lineage as vancomycin-resistant isolates previously isolated from Danish pigs.

Animals↗

PubMed2001

Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae.

The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two -10 boxes and a possible -35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both -10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.

Adhesins, Bacterial↗

PubMed2001

Monitoring of ascorbate at a constant rate in cell culture: effect on cell growth.

Ascorbic acid (vitamin C) is a primary antioxidant for cells. But, ascorbic acid added to culture medium is not readily available to cells in culture, because it is unstable in aqueous media. We determined the conditions required to obtain and maintain a constant concentration of ascorbate in the culture medium using ascorbate and ascorbate-phosphate. The study was carried out with human fibroblasts and the amounts of ascorbate in the culture medium were determined by high performance liquid chromatography. A mixture of 0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a constant concentration of ascorbate in the culture medium. This constant ascorbate concentration proved to be nontoxic for cells and stimulated cell growth in the short term and long term.

Ascorbic Acid↗

PubMed2001

Identification of a new mutation in the hepatocyte nuclear factor-1alpha gene in a Polish family with early-onset type 2 diabetes mellitus.

Recently, several genes associated with early-onset, autosomal dominant Type 2 diabetes (MODY) have been identified. Mutations in the hepatocyte nuclear factor (HNF)-1alpha gene seem to account for a substantial proportion of this type of diabetes in several populations. However, it is still of interest to estimate the frequency of HNF-1alpha mutations in various ethnic groups. The aim of our study was to determine the contribution of the HNF-1alpha gene to the development of MODY in a Polish population. We selected 15 families with MODY for this project. The 10 exons and promoter region of the gene were screened for sequence differences by direct sequencing of probands DNA. We detected 7 previously described polymorphisms that were not associated with diabetes. However, one sequence difference, a deletion of a cytosine in codon 225 in exon 3 (designated S225fdelC), was a new mutation resulting in a frame shift and synthesis of a nonsense peptide from amino acid 225 to 232 followed by the stop codon. Thus, the S225fdelC mutation effectively caused the loss of a part of the DNA binding domain and the entire transactivation domain. This mutation was present in 4 affected members of the family. They developed diabetes at an early age (mean age at diagnosis 23 yr) and were characterized by severely impaired insulin secretion. In addition, one family member who was not a carrier of the S225fdelC mutation was diagnosed with diabetes. Thus, he represents an example of phenocopy. In conclusion, we have identified a new HNF-1alpha variant that represents the first MODY mutation described in a Polish population. MODY3 mutations, including those in the exon 4 "hot spot", do not appear to be a very common cause of MODY in the Polish population.

Adolescent↗

PubMed2000

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system.

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.

Animals↗

PubMed2000

Epidemiological and microbiological study of an outbreak of infectious keratoconjunctivitis in sheep.

After several thousand sheep had been imported from Australia and New Zealand to Croatia during 1995, many native sheep that had been in contact with the imported animals acquired a severe ocular disease closely resembling infectious keratoconjunctivitis. In affected flocks glucose-fermenting mycoplasma were isolated from 48 per cent of conjunctival swabs and Branhamella ovis from 58 per cent. Twelve of 42 culturally and biochemically identical isolates were identified as Mycoplasma conjunctivae by polymerase chain reaction. From the conjunctivae of two animals M conjunctivae and M arginini were isolated in mixed culture. For many reasons most farmers removed the imported animals from their flocks and only sporadic cases of the disease were recognised in 1996. At the end of 1997, six flocks which were clinically free of the disease but had been affected during 1995, and five flocks with no history of the severe ocular disease were examined clinically and microbiologically, and were found to be free of M conjunctivae infection. At the time, B ovis was cultured almost exclusively from sheep originating from flocks which had been affected during 1995 and/or 1996. It was usually isolated in pure culture or as the predominant bacterial species, and was often accompanied by mild conjunctivitis. There were no microbiologically confirmed new cases of infectious keratoconjunctivitis during 1998 and 1999.

Animals↗

PubMed2000

Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies.

The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern.

ATP-Binding Cassette Transporters↗

PubMed2000

Apx toxins in Pasteurellaceae species from animals.

Pasteurellaceae species particularly of porcine origin which are closely related to Actinobacillus pleuropneumoniae were analyzed for the presence of analogues to the major A. pleuropneumoniae RTX toxin genes, apxICABD, apxIICA and apxIIICABD and for their expression. Actinobacillus suis contains both apxICABD(var.suis) and apxIICA(var. suis) operons and was shown to produce ApxI and ApxII toxin. Actinobacillus rossii contained the operons apxIICA(var.rossii) and apxIIICABD(var.rossii). However, only the toxin ApxII and not ApxIII could be detected in cultures of A. rossii. The Apx toxins found in A. suis and A. rossi may play a role in virulence of these pathogens. Actinobacillus lignieresii, which was included since it is phylogenetically very closely related to A. pleuropneumoniae, was found to contain a full apxICABD(var.lign.) operon which however lacks the -35 and -10 boxes in the promoter sequences. As expected from these results, no expression of ApxI was detected in A. lignieresii grown under standard culture conditions. Actinobacillus seminis, Actinobacillus equuli, Pasteurella aerogenes, Pasteurella multocida, Haemophilus parasuis, and also Mannheimia (Pasteurella) haemolytica, which is known to secrete leukotoxin, were all shown to be devoid of any of the apx toxin genes and did not produce ApxI, ApxII or ApxIII toxin proteins. However, proteins of slightly lower molecular mass than ApxI, ApxII and ApxIII which showed limited cross-reactions with monospecific, polyclonal anti-ApxI, anti-ApxII and anti-ApxIII were detected on immunoblot analysis of A. equuli, A. seminis and P. aerogenes. The presence of Apx toxins and proteins that imunologically cross react with Apx toxins in porcine Actinobacillus species other than A. pleuropneumoniae can be expected to interfere with serodiagnosis of porcine pleuropneumonia.

Amino Acid Sequence↗

PubMed2000

Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from Equidae.

Clostridium difficile is an antibiotic-associated emerging pathogen of humans and animals. Thus far three toxins of C. difficile have been described: an enterotoxin (ToxA), a cytotoxin (ToxB) and an ADP-ribosyltransferase (CDT). In the present work we describe the first isolation of CDT producing C. difficile from Equidae with gastro-intestinal disease. Out of 17 C. difficile strains isolated from Equidae, 11 were positive for the genes tcdA and tcdB encoding ToxA and ToxB. In addition four of these 11 isolates were positive for the cdtA gene encoding the catalytic subunit of the ADP-ribosyltransferase CDT. Interestingly none of the isolates derived from canines (41 isolates) and felines (4 isolates) harboured the cdtA gene. In C. difficile field isolates which contained the cdtA gene, ADP-ribosyltransferase activity could also be detected in culture supernatants indicating expression and secretion of CDT. All strains were associated with intestinal disorders, but no association was found for the occurrence of toxins with a specific clinical diagnosis.

ADP Ribose Transferases↗

PubMed2000

Isolation and characterization of a capsule-deficient mutant of Actinobacillus pleuropneumoniae serotype 1.

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroIpneumoniae, the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in the cpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly (P=0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.

Actinobacillus Infections↗

PubMed2000

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (betaGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of betaGP supply were tested. We compared 10 mM betaGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where betaGP was added at day 0. Furthermore, 10 mM betaGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Aged↗

PubMed2000

Practice of a testing bench to study the effects of cyclic stretching on osteoblast-orthopaedic ceramic interactions.

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.

Alkaline Phosphatase↗

PubMed2000

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing.

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Animals↗