Effects of antithrombin III on tumor necrosis factor-alpha and interleukin-1beta synthesis in vascular smooth muscle cells.

In the course of sepsis, severe coagulopathy and disseminated intravascular coagulation (DIC) are common events. Therefore, substances known to interfere with the coagulation cascade have been studied in animal models of sepsis. Among them, antithrombin III (AT III) was reported to be a promising therapeutic tool because it exhibited anti-inflammatory properties in addition to its anticoagulative effects. In our studies using vascular smooth muscle cells (VSMC) as a monoculture model, contradictory effects of AT III on the release of the proinflammatory agonists tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were found. Whereas AT III inhibited the lipopolysaccharide (LPS)-induced production of these cytokines on both the transcriptional and the translational levels when given at higher concentrations (5 or 10 U/ml), lower amounts of AT III did not show this suppressive effect. In contrast, 0.5, 1, and 5 U/ml AT III led to an enhancement of TNF-alpha synthesis when combined with LPS. To date, we cannot provide a mechanism to explain the AT III-promoted modulation of TNF-alpha and IL-1beta generation in VSMC. However, with respect to its potential therapeutic benefit in systemic inflammatory conditions, AT III should not be regarded strictly as an anti-inflammatory modulator.

Reevaluation of the incidence of thromboembolic complications in congenital factor XII deficiency--a study on 73 subjects from 14 Swiss families.

To further elucidate the debated role of hereditary FXII deficiency as a thrombophilic risk factor this follow-up study on 65 subjects out of 12 Swiss families was undertaken (follow-up: 6 yrs). Fifteen severely FXII deficient subjects (FXII:C < 1%), 35 partially FXII deficient subjects (FXII:C > or = 1-59%), 10 with normal FXII values (FXII:C > or = 70%), and 5 non-classifiable subjects (FXII:C > or = 60-69%) were reevaluated. Eight subjects (4 severely and 3 partially FXII deficient, 1 non-classifiable) were newly enrolled. Four instances of deep vein thrombosis, one superficial vein thrombosis and one myocardial infarction were noted in 2 out of 19 severely FXII deficient subjects during a total life-time period of 866.6 patient-years. In 38 partially FXII deficient subjects (1862.8 patient-years) one ischemic cerebrovascular stroke and one superficial vein thrombosis were recorded in 2 individuals. The 10 subjects with normal FXII values (498.2 patient-years) remained thrombosis-free. One superficial vein thrombosis occurred in an unclassifiable woman. None of the 3 different FXII gene defects revealed in our patients was specifically associated with thromboembolic complications. Kaplan-Meier analysis of thrombosis-free survival suggests that hereditary partial (and probably severe) FXII deficiency does not constitute a thrombophilic condition.

Direct gene diagnosis of cystic fibrosis by allele-specific polymerase chain reactions.

Cloning of the cystic fibrosis gene and the identification of the predominant disease-causing mutation did not only help in the understanding of this frequent disease, but was immediately followed by applications in direct gene diagnosis. We describe a method for the detection of the so-called delta F508 deletion, which accounts for 70% of the mutations: a polymerase chain reaction with two different combinations of oligonucleotide primers, which discriminate between mutant and wild-type alleles. This allele-specific amplification provides a rapid, non-radioactive and very reliable method for direct genotyping. Establishment of the procedure and its application in diagnosis are described. We further report preliminary data on the frequency of this mutation in German patients and its association with restriction fragment length polymorphism haplotypes.

Cloning, molecular characterization and chromosome localization of the inorganic pyrophosphatase (PPA) gene from S. cerevisiae.

The gene for Saccharomyces cerevisiae inorganic pyrophosphatase, PPA, has been cloned by hybridization of "long" oligonucleotide probes with both cDNA and genomic S. cerevisiae libraries. The nucleotide sequence of 1612 bp from a genomic subclone that includes the entire coding region gives a deduced amino acid sequence that has nine differences (out of a total of 286 residues) from the previously published amino acid sequence that was determined directly. The codon usage in PPA is as expected for a "highly expressed" yeast gene. The upstream region contains a poly dA/dT sequence that might comprise a constitutive promoter. The PPA gene appears to be present in a single copy within the S. cerevisiae genome and has been localized to chromosome II.

Homozygous factor V Leiden mutation in a woman with multiple adverse pregnancy outcomes.

We report a case with one intrauterine fetal death (IUFD) at 32 weeks of gestation, one premature delivery at the same week, and one abortion of unknown etiology at 12 weeks of gestation. We discuss that the presence of homozygosity for Factor V Leiden may be associated with placental insufficiency in this woman. Application of anticoagulant therapy may have been beneficial in her current pregnancy.

Detection of human spermatid-specific transcripts in peripheral blood lymphocytes of males and females.

We describe the detection of ectopic ("illegitimate") transcripts of the proacrosin and protamine 2 genes, which are specific for human spermatogenesis, in non-cultured peripheral blood lymphocytes. After specifically-primed reverse transcription of total lymphocyte RNA, these rare transcripts can be directly visualised after two rounds of polymerase chain reaction with nested primers. Sequence and restriction analyses of the corresponding fragments have confirmed that transcripts of proacrosin and protamine 2 are present in the lymphocytes not only of males, but also of adult females.

Two cystic fibrosis patients with the genotype G542X/G551D.

Two adult sisters affected by cystic fibrosis were both shown to carry two different alterations within exon 11 of the CFTR gene, the nonsense mutation G542X and the missense mutation G551D. Both patients exhibit a relatively benign clinical course. In the described patients, G542X functions as a "mild" allele and is, in this respect, dominant to the "severe" G551D.

A novel 5'-upstream mutation in the factor XII gene is associated with a TaqI restriction site in an Alu repeat in factor XII-deficient patients.

The factor XII gene from factor XII-deficient patients was screened for mutations at the genomic level. In patients negative for cross-reacting material, a T to C transition 224 bp upstream of exon 3 was identified (exon 3-224 (T --> C)) that creates an additional TaqI restriction site in intron B. This mutation is located within a putative hormone responsive element and within a B box promoter of an Alu repeat of the Sb0 family. The TaqI site is associated with a G to C transversion upstream of the transcription initiation site (exon 1-8 (G --> C)). We discuss the possible roles of these elements in factor XII gene regulation.

A cystic fibrosis patient homozygous for the nonsense mutation R553X.

A cystic fibrosis patient homozygous for the nonsense mutation R553X was identified by mutation screening and the genotype confirmed by DNA sequencing. This patient, the only one described to date who is homozygous for this stop codon in exon 11 of the CFTR gene, is moderately severely affected. Clinical and molecular findings are presented.

The effect of replication errors on the mismatch analysis of PCR-amplified DNA.

The mismatch analysis of PCR-amplified DNA has generally assumed the absence of artificially introduced base substitutions in a significant proportion of the amplification product. This technique, however, differs from the direct sequencing of amplified DNA in that non-specific substitutions will render a molecule useless in analysis. The expected signal-to-noise ratio is heavily influenced by several parameters viz. initial template copy number, number of replication cycles, eventual product yield and the type of experimental system adopted. Mathematical modelling can be used to optimize fragment length with respect to the method applied and suggests as yet undescribed improvements such as partial modification or cleavage to optimize signal detection.

A cystic fibrosis patient with the nonsense mutation G542X and the splice site mutation 1717-1.

A cystic fibrosis patient with the genotype G542X/1717-1 (G----A) was identified by DNA sequencing of exon 11 of the CFTR gene. The available molecular and clinical data are presented. This is the first report of a patient with this rare genotype and may serve to improve our understanding of allele interactions.

Nitric oxide-dependent apoptosis in ovarian carcinoma cell lines.

OBJECTIVE: In a recent study, we found different profiles of inducible nitric oxide synthase (iNOS) gene expression in the ovarian carcinoma cell lines OVCAR-3, HOC-7, and 2774 following stimulation by proinflammatory cytokines. The present study was performed to determine whether nitric oxide (NO) synthesis correlates with programmed cell death in these cells. METHODS: NO-Dependent apoptosis was detected by DNA fragmentation analysis and fluorescence-activated cell sorter analysis. RESULTS: NO formation in response to interferon gamma (IFN-gamma), interleukin-1beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha) was correlated with programmed cell death in the investigated cells. DNA fragmentation was most prominent in OVCAR-3 (34.17 +/- 1.81%), less pronounced in HOC-7 (12.86 +/- 0.45%), and undetectable in 2774 (4.54 +/- 0.40%) cells. The rate of apoptosis correlated with the amount of NO formation in cytokine-treated cells. Moreover, coincubation of OVCAR-3 and HOC-7 with the specific iNOS inhibitor aminoguanidine suppressed apoptosis induced by IFN-gamma, IL-1beta, and TNF-alpha. CONCLUSION: Our data indicate that in OVCAR-3 and HOC-7 cells, NO synthesis induced by IFN-gamma, IL-1beta, and TNF-alpha is correlated with the degree of apoptotic cell death. In clinical situations, this might in part explain the benefit of cytokine application in ovarian carcinoma patients (e.g., documented for IFN-gamma).

Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts.

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differential splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.

Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier.

Both normal and pathological transcripts of tissue-specific genes may be detected by polymerase chain reaction (PCR) amplification in tissues not normally considered to express the gene product. The exploitation of constitutive basal mRNA levels ("ectopic" transcription) would be a major boon to diagnostic medicine since it promises both to simplify the analysis of complex genes and to avoid the requirement for an expressing tissue that is sometimes obtainable only by biopsy. We have demonstrated the feasibility of this novel strategy by characterizing a mutation in the X-chromosomal Duchenne (or Becker) muscular dystrophy (DMD/BMD) gene encoding dystrophin. The massive size of this gene has in the past often hindered carrier detection due to the high frequency of recombination and the high proportion of new mutations. In this study a deletion was identified in both a BMD patient and a heterozygous carrier using only a minimal volume of peripheral blood. Following specifically primed reverse transcription of lymphocyte RNA, the relevant region of the pathological cDNA was PCR-amplified. Sequence analysis indicated an in-frame deletion of exons 45 to 47.

Mutations in the human factor XII gene.

The factor XII gene from 31 unrelated factor XII-deficient patients from Germany, Switzerland, and Austria was screened for mutations at the genomic level. Several novel mutations were detected and their absence in a control group of 74 healthy unrelated individuals was checked. Most changes are in the serine protease domain affecting the catalytic triad His-393-Asp-442-Ser-544; two missense mutations, R398Q (arginine 398 to glutamine; gene bank accession no. U71276) and L395M (leucine 395 to methionine; gene bank accession no. U71277), are close to the active site histidine at position 393. Another mutation detected in a cross-reacting material (CRM)-positive female with a history of three abortions affects the active site aspartic acid by changing it to asparagine (D442N; gene bank accession no. U71275). The novel mutation G570R (glycine 570 to arginine; gene bank accession no. U71274) giving rise to a CRM-positive phenotype is located next to Cys571, which forms a vital disulfide bridge. Two mutations are causing reading frame shifts: one single basepair deletion in exon 12 [exon 12: 10590(DelC); gene bank accession no. U71278] and one acceptor splice site mutation [exon 14: 11397(G --> A); gene bank accession no. L43615]. The putative regulatory mutation exon 1:-8 (g --> c) in the upstream region of the gene is associated with an aberrant Taq I restriction site allele in intron B of the gene (gene bank accession no. X80393).