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Biomedical subjects

D Wolf

Publications and source records attributed to D Wolf.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of a novel autosomal dominant bleeding disorder in a large kindred from east Texas.

A large east Texas family with autosomal dominant inheritance of a novel bleeding disorder has been identified. The disorder is characterized clinically by easy bruising, life-threatening bleeding with trauma or surgery, and menorrhagia in affected women. Laboratory studies demonstrated prolongation of the prothrombin time and activated partial thromboplastin time in affected individuals. Paradoxically, assays of known coagulation factors are all within normal limits. To determine the molecular basis of this disease, a candidate gene linkage analysis in this kindred was done. Initially it was hypothesized that the cause of the disease in this family could be an antithrombin III (AT3) mutation that resulted in a constitutively active AT3 in the absence of heparin binding. Linkage studies using DNA from the family and an intragenic polymorphic marker within the AT3 gene showed that the disease mapped to this locus. The coding region and intron/exon junctions of AT3 were sequenced using the proband's DNA, but this analysis failed to identify a mutation. Additional family members were recruited for the study, and 16 polymorphic markers around the AT3 gene were analyzed. Using 2 recombinants, the critical interval for the defective gene was narrowed to approximately 1.5 Mb, centromeric to AT3. The factor V (FV) gene was mapped into the disease interval and sequenced; there were no mutations found. Elucidation of the genetic defect causing the bleeding disorder in this family may reveal a novel protein involved in the coagulation cascade.

Adult↗
PubMed2001

TNF-alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1--relevance for concanavalin A-induced hepatitis.

TNF-alpha has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1(-/-) and TNFR2(-/-) mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-gamma. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1(-/-) and TNFR2(-/-) mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM(-/-) mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4(+) T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.

Animals↗
PubMed2000

Functional and structural defects in HIV type 1 nef genes derived from pediatric long-term survivors.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

Adolescent↗
PubMed2000

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes.

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.

Cell Nucleus↗
PubMed2000

[Transarterial oil chemoembolization for hepatocellular carcinoma, in 100 cases].

Hepatocellular carcinoma (HCC) is a common malignancy with a grave prognosis. Most patients have both the malignant tumor as well as hepatic cirrhosis. Liver transplantation or hepatectomy are considered the only curative procedures, but can be applied in fewer than 10% of patients. In recent decades the most common treatments of HCC are transarterial chemoembolization with oil (TOCE) and percutaneous ethanol injection (PEI). We summarize our retrospective study of 100 patients (mean age 64 +/- 3) treated by TOCE. In 271 procedures between 1989-1998, in 16 patients hepatectomy was combined with TOCE and in 8 PEI was combined with TOCE, while the rest were treated by TOCE alone. Tumor mass was reduced in 36% of those treated by TOCE (tumor volume reduced 24-75%). Alpha-feto protein (AFP) was reduced 25-90% in 20/32 of those with elevated AFP levels. Median survival for the 100 in the entire group was 19 months (10.9 months in those with conservative treatment). Median survival in the 57 in Okuda stage 1 and the 43 in stages 2 or 3 was 30.1 months and 10.9 months, respectively (p < 0.0001). Of the 57 in stage 1, 16 underwent hepatectomy in addition to TOCE and 41 were treated only by TOCE (median survival 15 and 26 months, respectively, p not significant). Comparing Okuda 1 patients treated by TOCE only with the natural history of the disease and historical controls (Okuda 1 patients treated conservatively in 1984) median survival was 26 and 10 months respectively (p < 0.001). The side effects of TOCE were relatively mild. There was 1 fatality (3 days after treatment), and quality of life was maintained. Despite progress in the treatment of HCC by TOCE, PEI, and liver transplantation, long-term survival has remained unsatisfactory.

Aged↗
PubMed2000

Importance of Kupffer cells for T-cell-dependent liver injury in mice.

T cells seem to be responsible for liver damage in any type of acute hepatitis. Nevertheless, the importance of Kupffer cells (KCs) for T-cell-dependent liver failure is unclear. Here we focus on the role of KCs and tumor necrosis factor (TNF) production after T cell stimulation in mice. T-cell- and TNF-dependent liver injury were induced either by Pseudomonas exotoxin A (PEA), by concanavalin A (Con A), or by the combination of subtoxic doses of PEA and the superantigen Staphylococcus enterotoxin B (SEB). KCs were depleted by clodronate liposomes. Although livers of PEA-treated mice contained foci of confluent necrosis and numerous apoptotic cells, hardly any apoptotic cells were observed in the livers of Con A-treated mice. Instead, large bridging necroses were visible. Elimination of KCs protected mice from PEA-, Con A-, or PEA/SEB-induced liver injury. In the absence of KCs, liver damage was restricted to a few small necrotic areas. KCs were the main source of TNF. Hepatic TNF mRNA and protein production were strongly attenuated because of KC-depletion whereas plasma TNF levels were unaltered. Our results suggest that KCs play an important role in T cell activation-induced liver injury by contributing TNF. Plasma TNF levels are poor diagnostic markers for the severity of TNF-dependent liver inflammation.

ADP Ribose Transferases↗
PubMed2000

Identification of a novel plant virus promoter using a potyvirus infectious clone.

A putative promoter from the strawberry vein banding caulimovirus (SVBV) genome was identified by its ability to drive infection with full-length cDNA of the zucchini yellow mosaic RNA potyvirus (ZYMV). A high rate of infection was obtained with the cDNA under control of the SVBV promoter using particle bombardment technology. The SVBV promoter shows 60% homology to the cauliflower mosaic virus 35S promoter in the domain spanning the conserved motifs of CCACT (at -83) and the TATA box (at -31), to the transcription start. The 3'-end one-third of the putative promoter (328 bp) was sufficient to invoke full infectivity with the ZYMV clone, and drove transient reporter gene expression in Solanaceae and Cucurbitaceae transformed with a binary plant transformation vector. Stable expression of a reporter gene (GUS) under control of the truncated SVBV promoter was shown in transformed tobacco shoots in roots, leaves and stems.

Base Sequence↗
PubMed2000

The relationship between fertility potential measurements on cryobanked semen and fecundity of sperm donors.

Sperm penetration assay (SPA) scores obtained from cryobanked semen were correlated with therapeutic insemination (TI) fecundity in a group of established sperm donors, thereby evaluating the efficacy of the SPA in screening donors for sperm banking. While the SPA has been used to separate fertile from infertile males, we altered assay conditions to use frozen semen and to distinguish performance among fertile donors. Three frozen ejaculates from 11 pregnancy-proven donors were analysed. Of 905 TI cycles, 275 recipients achieved 95 pregnancies. There were no significant relationships between fecundity and donor semen, washed sperm parameters, sperm recoveries or recipient age. A significant relationship was revealed between mean SPA scores (range 8.7-66.6 penetrations/ovum) and donor fecundity (range 0.04-0.16, P < 0.03). Sperm concentration was varied in an effort to establish the most sensitive test condition. Using 0.25x10(6) motile spermatozoa/ml, a highly significant relationship was observed (P < 0.002). The four donors with the lowest SPA scores achieved the four lowest fecundities. It is concluded that a modified SPA can be used on frozen donor semen to estimate donor fertility potential. If applied routinely in donor semen banking, poor quality applicants could be excluded, thereby increasing pregnancy rates while decreasing donor screening costs.

Adult↗
PubMed2000

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters postnatal development of seminal vesicle epithelium.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to alter male reproductive development of laboratory animals through in utero and lactational exposure. As a result of exposure, the accessory glands of the male reproductive tract, including the seminal vesicle, are decreased in size as determined by total weight of the tissue. Analysis of seminal vesicle weights over time suggests that the changes may be transient. Administration of 1.0 microg/kg TCDD during gestation caused a significant decrease in seminal vesicle weights of offspring 8-11 months of age. We examined the effects of TCDD on seminal vesicles from rats exposed in utero and lactationally. Pregnant Long Evans rats were gavaged on gestation day 15 with 1.0 microg/kg TCDD in corn oil. Male pups were euthanized and necropsied on postnatal days (PND) 15, 25, 32, 49, 63, and 120. Seminal vesicles were weighed and then fixed in 10% neutral buffered formalin and processed for microscopic examination. Seminal vesicle weights were not significantly decreased until PND 32. Androgen receptor mRNA expression in PND 25 seminal vesicles was not different from control. In the present study, TCDD exposure decreased seminal vesicle epithelial branching and differentiation. Control epithelial cells had tall columnar morphology with relatively abundant cytoplasm, whereas TCDD-treated cells had rounded nuclei and less cytoplasm. In addition, immunolocalization of proliferating nuclear antigen was confined to undifferentiated basal epithelial cells of controls but was found in both basal and luminal cells of the treated seminal vesicle. Results indicate that the TCDD-induced impaired growth of the rat seminal vesicles is associated with a dramatic decrease in the development of the epithelium.

Administration, Oral↗
PubMed1999

Comparison of the thermal denaturation behaviour of DNA-solutions and metaphase chromosome preparations in suspension.

Hyperchromicity measurements are well established to analyse the thermal denaturation behaviour of pure DNA sequences in solution. Here, we show that under appropriate experimental conditions this technique can also be applied to study thermally controlled conformation changes of higher order DNA-protein complexes as for instance metaphase chromosome preparations in suspension. A computer controlled sensitive, upright double beam photometer with a heatable cuvette was constructed. Measurements of the temperature dependent extinction of both, solutions and particle suspensions are possible, since sedimentation effects of particles can be neglected due to the vertical optical axis in the probe cuvette. Thermal denaturation of metaphase chromosome preparations of human and Chinese hamster cells was investigated and compared to melting profiles of DNA solutions for two excitation wavelengths, 256 and 313 nm. The influence of neutral and low pH was considered. The results indicate that metaphase chromosome preparations show a thermal denaturation behaviour different from pure DNA. Whereas DNA solutions showed one pH dependent melting peak at 256 nm only, the peak pattern of metaphase chromosome preparations showed a large variability both at 256 and 313 nm. At neutral pH, in two temperature regions (40-55 degrees C and 75-82 degrees C) peaks were found indicating chromosome typical conformation changes independently from the mammalian cell species (Chinese hamster, human). In contrast to pure DNA, no typical reduction in the temperatures of peak maxima with decreasing pH was found for metaphase chromosome preparations of both cell types. These results may be relevant for further systematic studies of efficient thermal probe/target denaturation procedures in non enzymatic DNA-chromosome in situ hybridisation.

Algorithms↗
PubMed1999

FtsK-dependent and -independent pathways of Xer site-specific recombination.

Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division. Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers. Two recombinases, XerC and XerD, act at the E. coli chromosomal recombination site, dif, and at related sites in plasmids. We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors. We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif. The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers. Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates.

Bacterial Proteins↗
PubMed1999

Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

Fas Ligand Protein↗
PubMed1999

Enteroaggregative and enterotoxigenic Escherichia coli among isolates from patients with diarrhea in Austria.

In a 3-month prospective study among 203 Austrian outpatients with diarrhea, the role of pathogenic Escherichia coli and the use of the polymerase chain reaction in screening Escherichia coli isolates from clinical stool specimens were evaluated. Enteroaggregative Escherichia coli and enterotoxigenic Escherichia coli combined were identified as the second most frequent cause of diarrhea. Of a total of 85 bacterial pathogens isolated from 80 patients, 15 were pathogenic Escherichia coli, 13 enteroaggregative Escherichia coli and two enterotoxigenic Escherichia coli. Enteropathogenic, enteroinvasive, and enterohemorrhagic Escherichia coli isolates were not detected.

Adolescent↗