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Biomedical subjects

A Schmitz

Publications and source records attributed to A Schmitz.

At least 91 records · Page 5Linked to original sources
PubMed1997

Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes.

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.

Amino Acid Sequence↗
PubMed1997

A cytogenetically anchored genetic map of bovine chromosome 1 obtained by integrating flow-sorted chromosome-derived microsatellite markers into the international bovine map.

A genomic library was constructed from a peak of flow-sorted bovine chromosomes 1 + X after PCR amplification. Forty-three bovine chromosome 1 microsatellites were isolated, genetically mapped and integrated in the international genetic map. In addition, BAC clones from a goat BAC library were identified for five markers (DVEPC119, INRA011, BM4307, KAP8 and MAF64). These goat BACs could be mapped by FISH onto bovine chromosome 1 to bands 1q44-->q45, 1q25, 1q21, 1q12 and 1q14-->q21, respectively. This map reduces the average interval between consecutive markers on the international bovine genetic map from 5.5 cM to 2.5 cM, and provides a good starting point for positional cloning projects in cattle, sheep or goats.

Animals↗
PubMed1997

[Localization of a tumor suppressor gene distal to D22S270 in colorectal cancers].

Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.

Adenocarcinoma↗
PubMed1997

[Autonomous thyroid adenoma. Value of thallium scintigraphy and pathology].

Clinical, biological, scintigraphic (99mTc, 123I, 201Tl) and histological data were reviewed in 90 patients operated for autonomous adenomas (hot nodules). Analysis of the data gave the following results: 74.4% of the nodules were solitary and 25.6% occurred in a multinodular goiter (MNG). Hyperthyroidism (FT3-, FT4-, TSH < 0.1) was present in 44% of solitary nodules and in 15% of MNG. 16.6% of patients were euthyroid, and 22.2% had a TSH < 0.1 and normal free hormones. The 201 Tl scintigraphy, performed instead of the administration of exogenous TSH, showed and uptake of Tl in the remainder of the thyroid. Four types of 201Tl uptake were observed in the pathological lobe: type 1: homogeneous uptake: type 2: heterogeneous uptake and hyperfixation in the nodule; type 3: faint uptake and hypofixation in the nodule; type 4: faint and very heterogeneous uptake. Histological analysis of the nodules showed 32.2% of follicular adenomas, 25.6% of MNG and 42.2% of colloid nodules. Among these lesions, 70% showed histological signs of hypermetabolism (resorption vesicles ...). Multivariate analysis of these data clearly demonstrates the need to redefine the clinical, biological, scintigraphic and histological concepts of autonomous adenomas. On the other hand, 91.6% of follicular adenomas show a type 2 Thallium uptake (22/24). On basis of these results, we prefer to operate these patients rather than to treat them by 131I or by percutaneous injection of ethanol.

Adenoma↗
PubMed1996

Myristoylation does not modulate the properties of MARCKS-related protein (MRP) in solution.

The members of the myristoylated alanine-rich C kinase substrate (MARCKS) family are proteins essential for brain development and phagocytosis. MARCKS proteins bind to actin filaments and calmodulin (CaM) and are phosphorylated by protein kinase C. In order to investigate how these interactions are regulated, we have characterized the properties of both the myristoylated (myr) and unmyristoylated (unmyr) forms of recombinant MARCKS-related protein (MRP), a 20-kDa member of the MARCKS family. Ultracentrifugation and circular dichroic spectroscopy reveal that MRP is an elongated protein, with an axis ratio estimated between 7 and 12 and with an apparent random coil conformation. MRP binds to CaM with high affinity (Kd,myr = 4 nM; Kd,unmyr = 7 nM) and with a second order rate constant, k+1,unmyr, of 1.6 x 10(8) M-1 s-1. In contrast to classical ligands such as the myosin light chain kinase, binding of MRP to CaM does not induce the formation of an alpha-helix in MRP. The catalytic subunit of protein kinase C (PKM) phosphorylates myr MRP with high affinity ([S]0.5 = 3.5 microM), positive cooperativity (nH = 2.5) and a turnover number of 130 min-1. CaM inhibits the phosphorylation of myr MRP with a half-maximum rate of phosphorylation at a [CaM]/[MRP] ratio of 0.7, indicating that CaM might efficiently regulate the phosphorylation of MRP in vivo. Interestingly, Ca2+ inhibits the binding of MRP to CaM as well as its phosphorylation by PKM in the millimolar concentration range, suggesting that MRP has a weak affinity for Ca2+. Finally, unmyr MRP can be stoichiometrically myristoylated by N-myristoyl transferase in vitro. Since neither binding of CaM nor phosphorylation by PKM inhibits myristoylation, the N terminus of unmyr MRP is exposed on the surface of the protein and is well separated from the effector domain. In view of the observations that unmyr and myr MRP do not exhibit significant differences in their properties in solution, the function of myristoylation is most probably to modulate the interactions of MRP with membranes.

Animals↗
PubMed1996

Human and porcine correspondence of chromosome segments using bidirectional chromosome painting.

The aim of this study was to determine the correspondence between human and porcine chromosome fragments using whole chromosome painting probes from both species in heterologous hybridization experiments (bidirectional heterologous chromosome painting). Bidirectional experiments allow the determination of segment-to-segment homologies between the chromosomes of these two species. Chromosome-specific painting probes from both species were, except one, obtained by DOP-PCR or PARM-PCR amplification of flow-sorted chromosomes. The probes labeled 95% of the total length of the porcine chromosomes with human painting probes and 60% of the human chromosomes in the reverse experiments. Syntenic relationships of chromosomal segments on the karyotype of both species were determined. There was close agreement between com- parative gene mapping data and the identified homologous segments; this comparison enabled orientation of the segments. We demonstrate that bidirectional heterologous chromosome painting is a highly efficient way of generating comparative cytogenetic maps.

Animals↗
PubMed1996

Lysozyme gene expression and regulation.

Analysis of lysozyme gene expression in chicken and mouse identified two evolutionarily different mechanisms of lysozyme gene regulation. The lysozyme gene in chicken is expressed in the oviduct and macrophage cells with expression regulated by different, partially overlapping sets of tissue specific cis-acting elements. In contrast to chicken, the mouse genome contains two lysozyme genes generated by a gene duplication event allowing each gene to be regulated by its own regulatory region. One gene is expressed in macrophages, the other in Paneth cells of the small intestine. The macrophage-specific gene contains a single strong enhancer in the 3'-flanking sequences that interacts with ubiquitously factors. Cytosine methylation of the core enhancer sequence has been implicated in the regulation of the enhancer activity. In spite of these evolutionary regulatory differences, the chicken lysozyme transgene retains macrophage-specific expression in mice.

Animals↗
PubMed1996

Homologous and heterologous FISH painting with PARM-PCR chromosome-specific probes in mammals.

Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30 degrees C during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.

Animals↗
PubMed1996

Neoepitope immunoassay: an assay for human interleukin 1 beta based on an antibody induced conformational change.

A secondary monoclonal antibody (mAb2) was generated by immunization with immune complexes of human IL-1 beta and a primary monoclonal (mAb1). mAb2 bound to a neoepitope on the IL-1 beta/mAb1-complex with a dissociation constant (Kd) of 26 pM but not to uncomplexed IL-1 beta. As assessed by the binding of labeled IL-1 beta and neutralization of bioactivity, mAb2 enhanced the affinity of IL-1 beta to mAb1; Kd-values were 108 pM in absence and 5.4 pM in presence of mAb2. By analyzing a series of mutants of IL-1 where surface loops had been exchanged with the corresponding loops of human IL-1 receptor antagonist protein, a critical region responsible for mAb2 binding was localized to the C-terminal region. In addition to mAb1/IL-1 beta-complexes, mAb2 bound pro-IL-1 beta/mAb1 complexes as well as pro-IL-1 beta suggesting that mAb2 recognized a conformation of IL-1 beta resembling that of pro-IL-1 beta. Using this pair of mAbs, chemiluminescent and enzyme linked assays with detection limits of 2 pg/ml hIL-1 beta have been established.

Animals↗
PubMed1996

Assignment of markers by using polymerase chain reaction on pools of swine flow-sorted chromosomes.

Gene chromosomal assignment can be realized not only by somatic hybrid panels but also by spot-blot hybridization or polymerase chain reaction (PCR) of flow-sorted chromosomes. We propose a swine chromosome assignment strategy by PCR amplification on pooled chromosomal DNA, which allows assignment despite possible chromosomal contamination during sorting. Each pool contains three different chromosomes, each chromosome being present in one or two pools. We present concordant results obtained for eight markers already mapped to different swine chromosomes and we assign the somatostatin gene to chromosome 13, a new marker in the pig genome.

Animals↗
PubMed1996

[Fulminant or sub-fulminant hepatocellular failure caused by metastatic invasiveness of the liver: an other cause of hypoxic hepatitis?].

OBJECTIVES: The purpose of this retrospective study was to report a series of 9 new cases of fulminant hepatic failure due to metastatic liver disease and to identify signs to support a theory of hypoxia. METHODS: In these 9 cases and in 38 previously published cases of fulminant or subfulminant liver failure, we looked for clinical (shock, sepsis, and cutaneous signs of circulatory failure), laboratory (marked increase in serum aminotransferases levels) and histological (cell necrosis) parameters compatible with liver hypoxia. RESULTS: Cutaneous signs of circulatory failure or shock were observed in 3 of the 9 cases in this study, and were not due to cardiogenic or septic shock. A marked increase in serum aminotransferases levels, 10 times above the upper limit of normal, was observed in 8 of the 9 cases in the present study and in 23 of 37 cases of the literature. Liver cell necrosis was observed in 6 of the 7 autopsied patients in this series and in 20 of 34 cases in the literature. Generally, liver cell necrosis was more severe than tumor cell necrosis and was related to the degree of sinusoidal involvement by tumoral cells. In one case, hepatic blood flow was decreased despite a high level of cardiac blood flow suggesting impairment of circulation in the liver. CONCLUSIONS: These 9 cases and a review of the literature support the hypothesis of hypoxic liver cell necrosis leading to acute liver failure in cases of metastatic liver disease. However, liver hypoxia cannot explain all the reported cases and was never due to circulatory failure from cardiac or septic causes, but to the sinusoidal involvement by tumor cells.

Adult↗
PubMed1996

In vivo protein interaction with the mouse M-lysozyme gene downstream enhancer correlates with demethylation and gene expression.

Differentiation of myeloid precursor cells results in transcriptional activation of the myeloid-specific murine M-lysozyme gene. M-lysozyme gene expression depends on the differentiation state of the myeloid cells and provides a marker for myeloid leukemias. The mouse lysozyme downstream enhancer (MLDE) was colocalized previously with the DNase I hypersensitive site in the chromatin of mature macrophages and shown to be macrophage differentiation-dependent. The correlation of the hypersensitive site appearance with expression of the M-lysozyme gene suggests that the enhancer becomes activated during macrophage differentiation. However, the predominant MLDE-binding protein GABP is ubiquitously expressed, indicating that additional regulatory mechanisms are required for restricting the tissue-specific activity of the enhancer. To demonstrate the specificity of the enhancer in vivo, we examined the in vivo interaction of factors with the MLDE in T cells, immature macrophage cells, and in macrophage cells. Although identical DNase I protection activity is present in extracts from all tested cell lines in vitro, the in vivo interaction of proteins is restricted to mature macrophage cells. The presence of factors capable of interacting with the enhancer is not sufficient for enhancer activity, suggesting that the process of differentiation results in factor accessibility for the MLDE. Analysis of the MLDE methylation state revealed a correlation between demethylation of the single CpG dinucleotide within the MLDE sequence and the in vivo interaction of proteins.

Animals↗
PubMed1996

[Liposarcoma of the spermatic cord].

The liposarcoma is mainly located in the retroperitoneum and rarely involves the spermatic cord. Dissemination occurs more often via haematogenous way than by lymphatic way. The incidence of local recurrence is higher than distant metastases. The treatment of choice is the wide excision. Adjuvant radiotherapy is recommended in the well differentiated and myxoid type of tumors if the local control during excision is not complete or wide enough. Benefit of chemotherapy is still controversial and limited. Case report of a stage 1 liposarcoma of the spermatic cord, four years after excision of a large lipoma during a hernia repair. The treatment consisted in radical excision of this liposarcoma without any adjuvant therapy. Follow-up of 30 months without recurrence.

Adult↗
PubMed1995

Methylation of the mouse M-lysozyme downstream enhancer inhibits heterotetrameric GABP binding.

Expression of the mouse M-lysozyme gene is a specific marker for the differentiation of macrophage/granulocyte cell lineages. Analysis of the mechanisms regulating M-lysozyme gene expression revealed an enhancer element in the 3'-flanking region of the gene, termed the M-lysozyme downstream enhancer (MLDE). Here we demonstrate that the nuclear factors binding to MLDE are present in all tested myeloid and non-myeloid mouse cell lines. Sequence analysis of MLDE identified two different sequences, CAGGAAGT and CCGGAAGT, which match the consensus binding sequences for proteins of the ets gene superfamily. The two sites are oriented palindromicly and separated by 10 bp. DMS/DEPC interference assays revealed different patterns of DNA-protein contacts on the two sites. Mutation of each consensus sequence leads to an individual change in protein binding in vitro. Despite these differences, both sequences are bound by GABP, forming a heterotetrameric complex. Tissue specificity is correlated with demethylation of a single CpG dinucleotide located in one of the two Ets motifs. This site when methylated inhibits GABP binding to both sequences in non-macrophage cell types.

Animals↗
PubMed1995

In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER.

The signal for degradation of proteins in the endoplasmic reticulum (ER) is thought to be the exposure of internal domains which are buried when the protein has adopted its correct conformation and which are also exposed in assembly intermediates. This raises the question of why the intermediates are not degraded. We developed a system based on the peroxidase-catalyzed iodination of tyrosine residues which continuously monitors the exposure of internal domains of proinsulin. In CHO cells this system discriminated between assembly intermediates of wild type (wt) proinsulin and misfolded proinsulin, as shown by the exclusive iodination of a misfolded mutant which was finally degraded in the ER. Iodination in vitro showed that the assembly intermediates of wt proinsulin also exposed internal domains. This iodination was inhibited by the addition of the molecular chaperone Bip which was co-immunoprecipitated with proinsulin in CHO cells. The results obtained with the mutant proinsulin support the assumption that exposed internal domains represent the signal for degradation in the ER. Observations of wt proinsulin show that Bip masks internal domains of normal assembly intermediates during the entire assembly process, thereby suppressing their degradation. We propose that internal domains contain co-localized signals for Bip binding and for degradation.

Adenosine Triphosphate↗