PubMed Health⌕ Search

Biomedical subjects

D Wolff

Publications and source records attributed to D Wolff.

At least 37 records · Page 2Linked to original sources
PubMed2000

Inositol 1,4,5-trisphosphate but not ryanodine-receptor agonists induces calcium release from rat liver Golgi apparatus membrane vesicles.

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP(3)) and ryanodine receptor agonists on Ca(2+) release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with (45)Ca(2+). Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP(3) at concentrations ranging from 100 nm to 100 microm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, promoted a rapid decrease in the Ca(2+) content of GA vesicles. The amount of Ca(2+) released from the vesicles was a function of IP(3) concentration, reaching about 60% in both GA and ER fractions at 100 microm IP(3). Calcium release was inhibited by heparin, an antagonist of IP(3) receptors. Calcium exhibited a bell-shaped effect on IP(3)-dependent Ca(2+) released from GA vesicles: it activated Ca(2+) release at concentrations up to 1 microm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca(2+) release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP(3)-sensitive Ca(2+) release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca(2+) release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane.

Adenosine Diphosphate Ribose↗
PubMed2000

Kinetic characterization of calcium uptake by the rat liver Golgi apparatus.

We carried out a kinetic characterization of the Ca(2+)active transport in the rat liver Golgi Apparatus (GA) membrane. Calcium accumulation by vesicles of a GA enriched fraction was found to be a function of both Ca(2+)and ATP-Mg concentrations, it was inhibited by 2 microm thapsigargin but not stimulated by 3 microm calmodulin. The kinetic parameter values obtained for the GA Ca(2+)pump were: J(max)of 3.96 nmol/mg min, K(m)for Ca(2+)of 0.150 microm and two K(m)'s for ATP of 1.14 microm and 519 microm. These results were almost identical to those obtained for the endoplasmic reticulum (ER) fraction, indicating that the GA Ca(2+)pump is a sarco/endoplasmic reticulum (SERCA) P-type, analogous-if not identical-to that present in the ER.

Adenosine Triphosphate↗
PubMed1998

Structure of the enzymatically synthesized fructan inulin.

Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60 x 10(6) and 90 x 10(6) g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples.

ATP-Binding Cassette Transporters↗
PubMed1998

Characterization of a supernumerary small marker X chromosome in two females with similar phenotypes.

We describe two female patients mosaic for a cell line with an extra marker X chromosome in addition to a normal 46,XX cell line. To our knowledge, these cases are the first reports of females who had a cell line with a supernumerary marker X chromosome in addition to a normal cell line. They also had strikingly similar manifestations, including small hands and feet, minor facial anomalies, obesity, and mental retardation. The DNA content of the mar(X) chromosomes was investigated by fluorescent in situ hybridization using pericentromeric probes. The XIST gene, which is necessary for initiation of X-inactivation, was deleted from both marker chromosomes, suggesting that these chromosomes were not subject to inactivation. The short arm breakpoints of the mar(X)s were between the DNA markers DXS423E on Xp11.21 and UBE1 on Xp11.23. In Patient 1, mar(X) contained the androgen receptor gene and the DNA marker DXS1, both mapping to Xq11.2, whereas in Patient 2 the chromosome breakpoint was proximal to these markers. We suggest that the similar phenotypes of these patients may be due to the overexpression of genes in the common pericentromeric region of the X chromosome.

Abnormalities, Multiple↗
PubMed1998

Second unrelated bone marrow transplantation without additional conditioning therapy after engraftment failure.

A 37-year-old female highly alloimmunized by multiple transfusions received a sex matched HLA-identical unrelated bone marrow transplant for hypoplastic MDS-RA with moderate myelofibrosis. Conditioning consisted of total body irradiation, cyclophosphamide and ATG, GVHD prophylaxis consisted of CsA, MTX and prednisolone. The CD34+ stem cell content of the first graft was relatively low due to an inadequate harvest. The patient appeared not to have engrafted by day 23 post-BMT. She therefore received a second sex mismatched HLA-identical unrelated bone marrow graft on day 25 after two days of 3.5 mg/kg methylprednisolone from a different donor. Over the ensuing days, the first marrow showed slow engraftment followed by engraftment of the second graft. The first graft was then rejected, as monitored by peripheral blood studies of chimerism. No signs of acute GVHD were observed. Despite successful trilineage engraftment and complete second donor chimerism, the patient died from disseminated toxoplasmosis encephalitis and pneumonia on day +104.

Adult↗
PubMed1996

Significance of human cytomegalovirus DNA detection in immunocompromised heart transplant patients.

Peripheral blood samples (n= 1240), obtained at variable intervals from 483 heart transplantation (HTx patients under immunosuppressive therapy, and blood samples (n=1013) obtained upon blood donation from 1013 healthy anti-human cytomegalovirus (HCMV) positive blood donors, were tested for HCMV DNA by nested polymerase chain reaction (PCR). The detection limit of the nested PCR was determined to be less than 10 copies of the plasmid pRR 47, containing the HCMV immediate early gene. HCMV DNA was detected in 79 of 483 HTx patient (17%). To the contrary' HCMV DNA was only detected in 1 of 1013 anti-HCMV positive, healthy blood donors (0.1%). This PCR positive donor had recently contracted a primary HCMV infection. The rate of HCMV PCR positive immunosuppressed HTx patients in our study was lower than the rate of HCMV PCR positive healthy blood donors in previous reports in the literature. Blood samples (269 from 117 HTx patients) were assayed for HCMV DNA in peripheral blood leukocytes, HCMV DNA in plasma, and HCMV tegument protein 65 kDa (pp 65 antigen). Three laboratory diagnostic patterns were observed and related to clinical findings: (1) HCMV DNA only in leukocytes was observed in 26 patients, 7 of whom had HCMV disease, 5 of whom had graft rejection, and 14 of whom had no specific symptoms; (2) HCMV DNA both in leukocytes and in plasma (viremia) was observed in 3 patients, who were all symptomatic with HCMV disease; (3) HCMV DNA in leukocytes and in plasma (viremia) and pp 65 antigen were observed in 13 patients, all of whom were symptomatic (10 patients had HCMV disease, and 3 patients had graft rejection). A similar sequence of diagnostic patterns was observed in all symptomatic HCMV infections and reactivations in this study: HCMV DNA appeared first in peripheral blood leukocytes, then also in plasma, followed by pp 65 antigen detectable in peripheral blood leukocytes. Upon clinical recovery, these findings disappeared in reverse order. However, HCMV DNA remained detectable in peripheral blood leukocytes for several weeks. The detection of HCMV DNA in the peripheral blood is an exception, not the rule, even in severely immunosuppressed HTx patients. It indicates a pathological condition, albeit without clinical symptoms in some patients, and it is the earliest signal of HCMV replication. Of 42 patients in whom HCMV DNA was initially detected only in peripheral blood leukocytes, 16 patients progressed into viremia. Thus, HCMV-specific PCR performed on nucleic acid extracts from lysed peripheral blood is an appropriate method for the monitoring of HCMV infections in immunosuppressed HTx patients.

Antibodies, Viral↗
PubMed1996

The high-conductance channels of yeast mitochondrial outer membranes: a planar bilayer study.

The high-conductance channels present in the outer membranes of wild-type and porin-less yeast mitochondria have been characterized electrophysiologically after incorporation in planar bilayer membranes. The most prominent activity was ascribed to a voltage-dependent, substate-rich, cationic channel which generally inactivated at potentials positive in the cis compartment, in agreement with the observations from patch-clamp experiments on porin-less mitoplasts. This channel has been identified as the so-called "peptide-sensitive channel" (PSC). We also observed similar channels displaying either no inactivation, or inactivation at both positive and negative potentials. These latter properties match those already described for mammalian and yeast PSC, respectively. These different behaviors are tentatively explained as arising from the presence, or lack of, peptides bound to the PSC. Very high conductances, apparently due to cooperative gating, were frequently displayed. In wild-type membranes, activity ascribable to the porin was also observed.

Electric Conductivity↗
PubMed1996

Longitudinal monitoring of latent and active human cytomegalovirus infections in peripheral blood of heart transplant recipients by single-tube nested RT-PCR.

PCR is a sensitive diagnostic tool for the detection of human cytomegalovirus (HCMV) DNA in the peripheral blood of immunosuppressed transplant recipients. However, its specificity as a prognostic marker for clinical disease is unclassified, because infections considered to be latent may be detected by this method. In order to diagnose active viral infections, we used reverse transcription-PCR (RT-PCR) to identify HCMV mRNA in blood. We developed a single-tube nested RT-PCR with preformed PCR mixtures embedded in a trehalose matrix. Blood samples of 48 heart transplant recipients were investigated for HCMV DNA. 8 patients detected to be HCMV DNA positive after transplantation were investigated in longitudinal monitoring for at least 6 months. HCMV mRNA was found in 5 patients who developed HCMV related symptoms during the period of RNA detection. There was no clear relation between the onset of DNA detection and the first demonstration of mRNA. In 2 patients HCMV DNA could be detected 74 and 81 days before the appearance of mRNA, suggesting long persistence until active infection is started. In 3 patients HCMV mRNA disappeared during or immediately after the end of ganciclovir therapy. In contrast, HCMV DNA was detectable continuously for prolonged periods after therapy, indicating that the persistence of HCMV DNA is not influenced by ganciclovir treatment. In summary, HCMV DNA detection seems to be a reliable early marker for the differentiation of persistent and active HCMV infections in immunosuppressed patients. Our data show that viral mRNA detection is probably a better predictor of the effectiveness of antiviral therapy than viral DNA detection.

Antibodies, Viral↗
PubMed1996

Treatment of chronic graft-versus-host disease with ultraviolet irradiation and psoralen (PUVA).

Chronic graft-versus-host disease (GVHD) remains a difficult clinical problem to treat and manage. We have reviewed our treatment of 40 patients treated at a single institution with PUVA (ultraviolet irradiation and psoralen) over a 14 year period. Thirty-five patients were treated for refractory chronic GVHD and five patients were treated at presentation of high-risk chronic GVHD. Overall, 31 of 40 patients improved on PUVA treatment. Sixteen patients achieved a complete response to PUVA added to their GVHD regimen. Four of the 15 partial responders had complete resolution of cutaneous GVHD but persistence of other systemic manifestations. The remaining partial responders had at least a 50% improvement in GVHD. We have also used PUVA with a glass fiber extension to treat intra-oral GVHD. PUVA is well tolerated with a high rate of response in the skin and mild side effects except for three patients who had therapy discontinued after phototoxicity (burn).

Adolescent↗
PubMed1995

The high-conductance channel of porin-less yeast mitochondria.

Patch-clamp and planar bilayer experiments on porin-less yeast mitochondria have allowed the characterization of a cationic channel activated at matrix-side positive (unphysiological) potentials. In voltage-pulse experiments, inactivation was a faster process than activation and the time constant for inactivation was more steeply dependent on voltage than the one for activation. The channel exhibited various conductance states whose occupancy depended on the applied transmembrane potential. In bilayer experiments, the presence of the pCOx-IV leader peptide induced fast gating in a voltage-dependent manner. A comparison with previously described activities suggests that the pore may coincide with the peptide-sensitive channel (PSC) (Thieffry et al. (1988) EMBO J. 7, 1449-1454) as well as with two other activities (Dihanich et al. (1989) Eur. J. Biochem. 181, 703-708; Tedeschi et al. (1987) J. Membr. Biol. 97, 21-29) assigned to the mitochondrial outer membrane. The possible relationship of this channel to the mitochondrial megachannel is discussed.

Ion Channels↗
PubMed1995

Pharmacokinetics of tiagabine as add-on therapy in patients taking enzyme-inducing antiepilepsy drugs.

The effect of hepatic enzyme-inducing antiepilepsy drugs (AEDs) on the clinical pharmacokinetics of tiagabine, a new AED, was studied in the steady-state condition. Patients with epilepsy entered this two-day study on a previously stable regimen of one to three enzyme-inducing drugs (phenytoin, phenobarbital, carbamazepine, and/or primidone) and tiagabine.HCl (24, 40, 56, or 80 mg daily). Patients were confined on both days, and serial blood samples were collected. Plasma tiagabine concentrations were determined by high-performance liquid chromatography; pharmacokinetic parameters were calculated using noncompartmental methods. Tiagabine pharmacokinetics were linear at all doses, as substantiated by the lack of significant differences among groups for dose-adjusted Cmax, Cmin, and AUC0-6. Some diurnal variation occurred, as evidenced by a statistically significant time effect for dose-adjusted AUC0-6. The effect was small, however, and possibly not clinically relevant. The harmonic mean half-lives of 3.8 to 4.9 h were remarkably constant across dosages and shorter than those of historical control subjects not taking enzyme-inducing AEDs suggesting that epilepsy patients not taking enzyme-inducing AEDs may require lower tiagabine.HCl doses to achieve the plasma levels observed in patients taking these drugs.

Adolescent↗
PubMed1994

Histomorphometric analysis of the repair of a segmental diaphyseal defect with ceramic and titanium fibermetal implants: effects of bone marrow.

We used a rat femoral diaphyseal defect/implant model to quantify the ingrowth of bone, cartilage, and fibrous connective tissue in a comparative study of woven sintered titanium fibermetal and porous hydroxyapatite/tricalcium phosphate ceramic implanted with and without the addition of syngeneic bone marrow cells. The patterns of tissue growth into the implants were analyzed with respect to time, type of implant, and the presence or absence of syngeneic marrow. Significantly more bone was found in ceramic implants than in fibermetal implants, with the addition of syngeneic marrow than without it, and at 4 months than at 2 months. Significantly more bone was found at both time periods in ceramic implants with bone marrow than in any other combination studied. We hypothesize that these findings resulted from interactions between the implanted material and its surroundings, specifically its ability to serve as a substratum for cell attachment, and cells in and around the defect, whether surgically implanted or arising from the soft-tissue bed.

Animals↗
PubMed1994

Reduced levels of IE2 gene expression and shutdown of early and late viral genes during latent infection of the glioblastoma cell line U138-MG with selectable recombinants of human cytomegalovirus.

To establish stable culture conditions which support persistence of the human cytomegalovirus (HCMV) genome in a latent state, the expression of the bacterial neomycin phosphotransferase (neo) from HCMV recombinants was used for selection. Different cell lines were infected with HCMV recombinants. The human glioblastoma line U138-MG was rendered resistant to G418 and retained the viral genome. More than 90% of the cells expressed the viral IE1 protein of 72 kDa for a culture period of 18 months. Many fewer cells expressed IE2-encoded proteins. No late gene expression or infectious virus was detectable. IE2 gene expression in latently infected cells appeared to be restricted at the level of RNA accumulation. Treatment with TPA or retinoic acid led to enhanced expression of the IE2 gene and the early genes encoding pp65 (UL83) and p52 (UL44). Superinfection with wild-type HCMV led to replication of neo-recombinant virus, indicating that replication-competent virus had been retained in latently infected U138-MG and that the cells had kept their permissive phenotype. Latent HCMV infection in U138-MG cells provides a useful model system for studying the role of particular viral and cellular genes in latent and permissive infections.

Cytomegalovirus↗
PubMed1994

Hip arthroplasty in mentally impaired patients.

The outcome of elective hip arthroplasty in 11 mentally impaired patients was compared with a cohort of 244 mentally competent patients. The impaired patients had a diagnosis of cerebral palsy, schizophrenia, or Down's syndrome, and were unable to give their own surgical consent. Major complications requiring additional surgery occurred in 5 of the mentally impaired patients. Three patients sustained fractures of the femur below the stem and were treated with plating, 1 developed a deep infection requiring prosthesis removal, and 1 developed a thoracic decubiti with bone involvement requiring debridement and rotation flap coverage. Three of these patients also developed urinary tract infections. Of the other 6 mentally impaired patients, 3 developed urinary tract infections, and 1 had a deep venous thrombosis. Functional independence was improved after surgery in the mentally impaired patients. However, the average cost of hospitalization, length of stay, and complication rate were significantly higher (p < 0.05) than those of the mentally competent patients.

Activities of Daily Living↗
PubMed1993

Characterization of a small supernumerary ring X chromosome by fluorescence in situ hybridization.

We report on a male with mild learning disabilities who has a supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridization as a ring X chromosome with breakpoints in the juxacentromeric region. Replication studies suggest that the ring X is late-replicating. However XIST, a gene in the X inactivation centre interval which is expressed exclusively from the inactive X chromosome, is not present on the marker, nor is it expressed in the patient's cells. These results are discussed with respect to karyotype-phenotype correlations and X inactivation.

Base Sequence↗
PubMed1993

Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene.

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1-5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.

Blotting, Southern↗