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T Dunn

Publications and source records attributed to T Dunn.

At least 37 records · Page 2Linked to original sources
PubMed1998

A prospective study of the predictive value of polymerase chain reaction assay for cytomegalovirus in asymptomatic kidney transplant recipients.

Cytomegalovirus (CMV) infection carries the potential for high morbidity in transplant recipients. The institution of pre-emptive therapy prior to the onset of clinical disease on the basis of CMV-polymerase chain reaction (PCR) is very attractive. We prospectively studied 52 asymptomatic kidney transplant recipients to test the hypothesis that serial CMV-PCR assays during the first 3 months post-transplant would identify patients at risk for CMV disease. Twenty-three patients (44.2%) had positive CMV-PCR tests at least once; 2 (8.6%) developed CMV. None of the 29 patients continuously negative for CMV-PCR developed CMV disease. CMV-PCR status did not influence patient and graft survival or the incidence of acute rejection. We conclude that while a substantial number of kidney transplant recipients become positive for CMV-PCR in the early post-transplant period, only a minority will develop CMV disease. Negative CMV-PCR assay is an accurate negative predictor for CMV disease but the value of CMV-PCR as a guide for pre-emptive anti-CMV therapy in kidney transplant recipients appears limited.

Adult↗
PubMed1997

Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p.

The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Delta mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation.

Calcium↗
PubMed1997

Activation of divalent cation influx into S. cerevisiae cells by hypotonic downshift.

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells form YPD medium containing 0.8 M sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca2+ and other divalent cations into the cell. For cells grown in YPD at 37 degrees C, this hypotonic downshift increases Ca2+ accumulation 6.7-fold. Hypotonic downshift-induced Ca2+ accumulation and steady-state Ca2+ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg2+. The Ca(2+)-influx pathway responsible for hypotonic-induced Ca2+ influx may account for about 10-35% of Ca2+ accumulation by cells growing in YPD. Ca2+ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mM Cd2+ suggesting that mutants resistant to acute Cd2+ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx.

Amines↗
PubMed1997

Serine palmitoyltransferase (scs1/lcb2) mutants have elevated copy number of the L-A dsRNA virus.

The microsomal fraction isolated form serine palmitoyltransferase (lcb2/scs1) mutants is enriched in a 90 kDa protein. The protein was identified as the major coat (Gag) protein of the L-A dsRNA virus particles by partial sequencing and by its interaction with anti-Gag antibodies. The total amount of Gag in whole-cell lysates of scs1/lcb2 mutant cells is greater than in wild-type lysates indicating that the enrichment of the protein in the microsomal fraction of scs1/lcb2 mutant cells may result from increased copy number of the L-A dsRNA virus. This is supported by the findings that the mutants also have increased levels of L-A dsRNA. Altered sphingolipid synthesis in the scs1 mutant cells appears to increase the copy number of the L-A viral particles.

Acyltransferases↗
PubMed1997

Regulation of cellular Mg2+ by Saccharomyces cerevisiae.

Regulation of cellular Mg2+ by S. cerevisiae was investigated. The minimal concentration of Mg2+ that results in optimal growth of S. cerevisiae is about 30 microM and a half-maximum growth rate is attained at about 5 microM Mg2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg2+ across the plasma membrane would provide sufficient cytosolic Mg2+ (0.1-1 mM). The total cellular Mg2+ of cells grown in synthetic medium containing 1 mM Mg2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg2+ decreases to about 80 nmol/mg protein as the Mg2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg2+. Cells shifted into Mg(2+)-free medium continue to grow by utilizing the vacuolar Mg2+ stores. Mg(2+)-starved cells replenish vacuolar Mg2+ stores with a halftime of 30 min. following the addition of 1 mM Mg2+ to the growth medium. The data indicate that cytosolic Mg2+ is maintained by the regulation of Mg2+ fluxes across both the vacuolar and plasma membranes.

Biological Transport↗
PubMed1997

Analysis of the MHC class I antigen presentation machinery in human embryonal carcinomas: evidence for deficiencies in TAP, LMP and MHC class I expression and their upregulation by IFN-gamma.

The expression of the major histocompatibility complex (MHC) class I antigens is suppressed in early post-implantation embryonic cells as well as in embryonal carcinoma (EC) cells, but could be upregulated by treatment with interferon (IFN)-gamma or retinoic acid. In a number of human and murine tumours, defects in the expression of the different components of the MHC class I antigen processing machinery, such as the proteasomal subunits LMP-2 and LMP-7 and the peptide transporters TAP-1 and TAP-2, account for impaired MHC class I surface expression. Here, we analysed the constitutive and IFN-gamma regulated mRNA and protein expression of the LMP, TAP and MHC class I molecules in the human EC line 577LM. In comparison to lymphoblastoid control cells, poor constitutive mRNA and protein expression of LMP-7, TAP-1, HLA class I, and beta 2-microglobulin, but not of TAP-2 and LMP-2, was detected in 577LM cells. The lack of MHC class I surface expression on 577LM cells could not be enhanced either by culturing cells at low temperature or by their incubation with exogenous MHC class I specific binding peptides: thus, the defective MHC class I surface expression was not only caused by impaired generation and processing of antigenic peptides. IFN-gamma treatment of 577LM resulted in a significant increase of MHC class I surface expression which was preceded by an upregulation of TAP, LMP and MHC class I transcripts as well as of TAP-1 and TAP-2, but not of LMP-2 and LMP-7, protein expression. These data suggest that human EC cell lines show a stable expression of a MHC class I low/deficient phenotype. The deficiencies associated with this phenotype involve different levels of the MHC class I restricted antigen presentation machinery and could be modified by treatment with IFN-gamma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Competitive inhibition of photoaffinity labelling of P-glycoprotein by anticancer drugs and modulators including S9788.

The affinity of the multidrug resistance modulator S9788 to interact with P-glycoprotein was characterized by its ability to inhibit the photoaffinity labelling of plasma membranes of multidrug resistant chinese hamster ovary B30 cells by iodomycin. This iodinated analogue of daunomycin specifically photolabels P-glycoprotein in membrane vesicles as well as in intact cells. The multidrug resistance reversing agents verapamil and cyclosporin and the cytotoxic drugs vinblastine and daunomycin which are known to be recognized by P-glycoprotein competed with iodomycin for its binding site on P-glycoprotein. Vinblastine and cyclosporin bound with high affinity, S9788 and verapamil with medium affinity to P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression of stem-cell factor and its receptor c-kit protein in normal testicular tissue and malignant germ-cell tumours.

The proto-oncogene c-kit and its ligand stem-cell factor (SCF) may play an important role in the development of normal and malignant testicular tissue. This study investigates the presence of SCF and c-kit protein in 32 orchiectomy specimens of patients with testicular cancer, in 5 specimens of normal testicular tissue and in three established non-seminomatous germ-cell cancer cell lines (H12.1, H32, 577ML) by an immunohistochemical approach. Out of 9 testicular cancer specimens classified as pure seminomas, 7 (78%) showed a strong immunohistochemical reaction for both SCF and c-kit protein on the surface of the tumour cells. Fourteen non-seminomatous germ-cell tumours composed of embryonal carcinoma were completely negative for both SCF and c-kit proteins and only faint positivity was found in 6 tumours (26%). Differentiated teratomatous structures within the specimens on non-seminomatous tumours showed a strong immunohistochemical reaction for SCF and c-kit protein in 8 of 11 (73%) cases. All three testicular cancer cell lines showed only faint staining reactions for c-kit protein and none for SCF. No secretion of SCF by the three lines in vitro was detected. The addition of high concentrations of SCF (100 ng/ml) to the testicular cancer cell lines in culture conditions without fetal calf serum resulted in a 1.4 to 3-fold growth stimulation compared to cell growth in serum-free medium alone. This effect was not detectable when the cells were cultured in serum-containing media. In the normal testicular tissue the germ-cells displayed a strong immunohistochemical reaction for c-kit protein while SCF positivity was found at the tubular membrane and on the surface of Sertoli cells. The SCF/c-kit system may possess a regulatory function in normal testicular tissue by possibly providing the microenvironment necessary for spermatogenesis. With the development of testicular cancer, this regulatory system seems to be lost, particularly in non-seminomatous germ-cell tumours. A growth-stimulatory effect of high concentrations of SCF on non-seminomatous testicular cancer cell lines can be detected only in culture conditions with serum-free media. The effects achievable by the combination of SCF with other growth factors need to be further studied, as well as the role of the c-kit/SCF regulatory system for normal spermatogenesis and its possible implications for the understanding and treatment of male infertility.

Germinoma↗
PubMed1996

Silibinin protects against cisplatin-induced nephrotoxicity without compromising cisplatin or ifosfamide anti-tumour activity.

Cisplatin is one of the most active cytotoxic agents in the treatment of testicular cancer, but its clinical use is associated with side-effects such as ototoxicity, neurotoxicity and nephrotoxicity. Long-term kidney damage from cisplatin particularly affects the proximal tubular apparatus and can be detected by increased urinary excretion of brush-border enzymes, such as L-alanine-aminopeptidase (AAP), and magnesium. In the current study, the flavonoid silibinin was used as a nephroprotectant for cisplatin-induced nephropathy in a rat animal model. Infusion of silibinin before cisplatin results in a significant decrease in glomerular (indicated by creatinine clearance and serum urea level) and tubular kidney toxicity (excretion of brush-border enzymes and magnesium). Silibinin given alone had no effect on renal function. In order to exclude an inhibition of the anti-tumour activity of cisplatin and 4-hydroperoxy-ifosfamide by co-administration of silibinin, in vitro studies were performed in three established human testicular cancer cell lines. Dose-response curves for cisplatin (3-30 000 nmol) combined with non-toxic silibinin doses (7.25 x 10(-6) or 7.25 x 10(-5) mol l-1) did not deviate significantly from those of cisplatin alone as measured by relative cell survival during a 5 day assay using the sulphorhodamine-B staining technique. Also silibinin did not influence the cytotoxic activity of 4-hydroperoxy-ifosfamide (30-10 000 nmol) in vitro. In summary, these in vitro data rule out a significant inhibition of the anti-tumour activity of the major nephrotoxic components, cisplatin and 4-hydroperoxy-ifosfamide, by co-administration of silibinin in a human germ cell tumour cell line model. Together with these demonstrated cytoprotection effects in the rat animal model, these data form the basis for a randomised clinical trial of silibinin for the protection of cisplatin-associated nephrotoxicity in patients with testicular cancer.

Animals↗
PubMed1995

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

Adenosine Triphosphatases↗
PubMed1994

Suppressors of the Ca(2+)-sensitive yeast mutant (csg2) identify genes involved in sphingolipid biosynthesis. Cloning and characterization of SCS1, a gene required for serine palmitoyltransferase activity.

Suppressor mutations in Saccharomyces cerevisiae that block Ca(2+)-induced death of csg2 mutant cells were investigated. These mutants, called scs mutants (suppressor of Ca2+ sensitivity), fall into seven complementation groups (scs1-scs7). All mutant strains in two of the complementation groups (scs1 and scs2) simultaneously acquire a requirement for 10 mM Ca2+, whereas wild type grow with only trace amounts of Ca2+. SCS1 was cloned by complementation of its Ca(2+)-requiring phenotype and found to be homologous to a family of pyridoxal phosphate enzymes that catalyze acyltransfer reactions. Secondary phenotypes of the scs1 mutants indicate that SCS1 is required for serine palmitoyltransferase activity which catalyzes the first committed step in sphingolipid biosynthesis (palmitoyl-CoA + serine-->3-ketosphinganine+CoASH+CO2). Other scs mutants as well as the csg2 null mutant have altered sphingolipid metabolism. The data suggest that sphingolipid metabolism in yeast is either regulated by Ca2+ and/or is required for Ca2+ homeostasis.

Acyltransferases↗
PubMed1994

Regulation of cellular Ca2+ by yeast vacuoles.

The role of vacuolar Ca2+ transport systems in regulating cellular Ca2+ was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate. The magnitude of the Ca2+ gradient generated by the vacuolar H+ gradient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentration of 125 nM would give a vacuolar free Ca2+ concentration of approximately 30 microM. The total vacuolar Ca2+ concentration is approximately 2 mM due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate from the vacuole is less than the growth rate indicating that the steady-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by growth. Based on the kinetic parameters of vacuolar Ca2+ accumulation in vitro, the maximum rate of Ca2+ accumulation in vivo is expected to be approximately 0.2 nmol of Ca2+ min-1 mg protein-1, a rate that is similar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concentration increases from 0.1 microM to 1-2 microM as the extracellular Ca2+ concentration is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg by increasing the rate of vacuolar Ca2+ accumulation but does not significantly alter the cellular growth rate.

Biological Transport↗
PubMed1994

A novel protein, CSG2p, is required for Ca2+ regulation in Saccharomyces cerevisiae.

Nineteen mutants that lost the ability to grow in 100 mM Ca2+ (but remained insensitive to 50 mM Sr2+) were identified in a screen of approximately 60,000 mutagenized yeast colonies. Cells carrying mutations in the CSG2 gene grow normally in low Ca2+ medium but have decreased growth rates when the Ca2+ concentration is above 10 mM. The csg2 mutant cells accumulate much higher levels of Ca2+ in a compartment that is exchangeable with extracellular Ca2+ but the nonexchangeable Ca2+ pool which predominates in wild-type cells is not influenced. Sr2+ influx is not increased in the csg2 mutant cells. Mg2+ decreases the amount of Ca2+ in the non-exchangeable pool without influencing the csg2-induced exchangeable Ca2+ pool. The data indicate that the csg2 mutation causes a selective increase in Ca2+ accumulation into a pool which is distinct from the vacuolar pool. The CSG2 protein consists of 410 amino acids, contains nine putative transmembrane segments, four potential sites for N-linked glycosylation, and a sequence with homology to the EF-hand Ca(2+)-binding site.

Amino Acid Sequence↗
PubMed1994

Modulation of cytostatic drugs by nifedipine in two heterotransplanted human testicular-cancer cell lines differing in their sensitivity to standard agents.

Drug resistance is an important clinical problem in testicular cancer patients with relapsed or refractory disease after first-line chemotherapy. Here we report that the relative reduction in tumour volume in nude mice heterotransplanted with either H 12.1 or H 23.1 human testicular cancer cell lines was significantly increased by addition of the calcium antagonist nifedipine to the maximum tolerated dose (MTD) of cisplatin (DDP). The mean reduction in relative tumour volume at day 30 (rVR) reached statistical significance for both cell lines following combination therapy of DDP with nifedipine compared to DDP alone (55 +/- 7% versus 12 +/- 4% for H 23.1 and 60 +/- 9% vs. DDP with nifedipine has also been confirmed in H 12.1 cells in vitro. However, in vivo, this combination was associated with a concordant increase in therapeutic toxicity. In contrast, no improvement in in vivo anti-tumour activity was obtained by combining similar dose-schedules of nifedipine with the MTD of epirubicin, or with MTDs of vinblastine or etoposide. These results are in agreement with our immunohistochemical finding that H 12.1 and H 23.1 do not over-express the Pgp 170 glycoprotein which mediates the multiple drug resistance (MDR) phenotype and involves both anthracyclines and vinblastine, but not DDP. We conclude that another Pgp-MDR modulator, nifedipine, is able to increase the anti-tumour activity of DDP in vivo and in vitro via a specific but as yet unknown mechanism, which is most likely not MDR-related. However, the increased anti-tumour activity is, in vivo, associated with a considerable increase in overall toxicity. Further studies are necessary to decrease therapeutic toxicity, before clinically relevant models for modifiers of DDP-resistance could possibly be applied to patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Sequence, mapping and disruption of CCC1, a gene that cross-complements the Ca(2+)-sensitive phenotype of csg1 mutants.

We have isolated, sequenced, mapped and disrupted a novel gene, CCC1, from Saccharomyces cerevisiae. This gene displays non-allelic complementation of the Ca(2+)-sensitive phenotype conferred by the csg1 mutation. The ability of this gene, in two copies per cell, to reverse the csg1 defect suggests it may have a role in regulating Ca2+ homeostasis. The sequence of CCC1 indicates that it encodes a 322 amino acid, membrane-associated protein. The CCC1 gene is located on the right arm of chromosome XII.

Amino Acid Sequence↗
PubMed1994

ERG gene is translocated in an Ewing's sarcoma cell line.

Ewing's sarcoma (ES) and related neoplasias are characterized by the reciprocal translocation, t(11;22)(q24;q12). The translocation has been reported to generate a fusion gene between the EWS (a previously undescribed gene on chromosome 22) and FLI1 genes. We report a similar translocation of EWS and FLI1 in an Askin's tumor cell line (SK-NM-C). Further, we describe an alternative translocation in an ES cell line (#5838) in which the 5' end of the EWS gene is juxtaposed to the 3' end of the ERG gene. The ERG gene is on chromosome 21, but no microscopically visible changes in chromosome 21 were observed. Elevated steady state levels of the EWS/ERG fusion gene transcript were detected in the #5838 cell line. This is the first report of a structural alteration of ERG in human cancer. Also, it confirms a general mechanism of generating putative oncogenic fusion genes by placing an ETS DNA binding domain in direct proximity to the carboxy terminus domain (CTD) related region of the EWS gene.

Base Sequence↗