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Biomedical subjects

T Wagner

Publications and source records attributed to T Wagner.

At least 181 records · Page 10Linked to original sources
PubMed1994

Cystamine potently suppresses in vitro HIV replication in acutely and chronically infected human cells.

We have investigated the effects of cystamine on the replication of human immunodeficiency virus (HIV) in human lymphocytes and macrophages, the natural targets of HIV in vivo. Treatment of chronically infected macrophages with cystamine, at a concentration (500 microM) that did not show any cytotoxic or cytostatic effects, strongly decreased (> 80%) HIV-p24 antigen production and completely abolished the production of infectious viral particles. Cystamine does not affect viral transcription, translation or protein processing; indeed, all HIV proteins are present in a pattern similar to that of nontreated cells. Instead, cystamine interferes with the orderly assembly of HIV virions, as shown by electron microscopy analysis, that reveals only defective viral particles in treated cells. Moreover, suppression of HIV replication, due to the inhibition of proviral DNA formation was observed in acutely infected lymphocytes and macrophages pretreated with cystamine. These results show that cystamine potently suppresses HIV replication in human cells by contemporaneously blocking at least two independent steps of the viral life cycle, without affecting cell viability, suggesting that this compound may represent a new possibility towards the treatment of HIV-1 infection.

Antiviral Agents↗
PubMed1994

Development of neuroepithelial tumors of the adrenal medulla in transgenic mice expressing a mouse hypothalamic growth hormone-releasing hormone promoter-simian virus-40 T-antigen fusion gene.

Expression of the mouse GH-releasing hormone (GRH) gene is restricted to neurons within the hypothalamus and to placenta. In an attempt to generate immortalized mouse hypothalamic neurons expressing GRH, the proximal 872-nucleotide segment of the 5'-flanking region of the hypothalamic mouse GRH gene was cloned by polymerase chain reaction and ligated to a 2.7-kilobase DNA sequence encoding the simian virus-40 (SV40) T-antigen, so that regulation of SV40 T-antigen expression was dependent on sequences within the mGRH 5'-flanking region. This region contains both TATA and CAAT boxes. The mouse GRH/SV40 T-antigen fusion gene was injected into 1-cell mouse embryos, and SV40 T-antigen incorporation in the mouse genome was found in 11 of 77 live births (3 males and 8 females). Although no evidence of hypothalamic tumors was found, all mice that expressed the transgene also developed tumors originating in the adrenal medulla. Gene copy number varied from 1-20 and was inversely proportional to survival, which ranged from 7-16 weeks. Corticosterone levels were normal. The male transgenic mice were fertile, and their progeny expressed the transgene and developed similar tumors. Microscopic examination of the tumors revealed a primitive neuroectodermal neoplasm that exhibited hematogenous and lymph node metastases and contained 100 ng norepinephrine, 2.85 ng epinephrine, and 1.1 ng dopamine/mg tumor tissue. Primary culture of dispersed tumor cells released norepinephrine into the medium (180 pg/ml.24 h). Cell lines from 2 tumors were established and exhibited characteristics similar to those of mixed neuroblastoma or primitive neuroectodermal tumors. In conclusion, the proximal 872 nucleotides of the hypothalamic mouse GRH promoter contain elements directing tissue-specific expression limited to early adrenal neuroectodermal cells. Other GRH DNA sequences appear to be required for restricted expression of mouse GRH within the hypothalamus.

Adrenal Gland Neoplasms↗
PubMed1994

The proximal promoter of the bovine luteinizing hormone beta-subunit gene confers gonadotrope-specific expression and regulation by gonadotropin-releasing hormone, testosterone, and 17 beta-estradiol in transgenic mice.

Transient transfection studies have proven useful in unraveling the molecular mechanisms underlying gonadotrope-specific expression and hormonal regulation of the gene encoding the alpha-subunit of the glycoprotein hormones. In contrast, similar studies performed with the LH beta gene have been less informative. When assayed by transient transfection into alpha T3-1 cells, activity of a 776-basepair bovine LH beta promoter-chloramphenicol acetyltransferase fusion gene (bLH beta CAT) was no greater than that of a promoterless control. To determine whether limited activity in vitro reflected the absence of critical regulatory elements, we examined activity of bovine LH beta fusion genes after stable integration in transgenic mice. In contrast to transient transfection studies, the LH beta promoter targeted high levels of CAT expression specifically to the pituitary. In addition, a bLH beta TK fusion gene was active only in gonadotropes. The bLH beta CAT transgene was also evaluated for responsiveness to gonadal steroids and GnRH. Testosterone and 17 beta-estradiol were capable of suppressing activity 70-80% in castrated males, despite the absence of high affinity binding sites for androgen or estrogen receptors. This suggests that feedback inhibition of LH beta CAT transgene expression by gonadal steroids may occur through an indirect mechanism, possibly at the level of the hypothalamus. To address whether the bLH beta CAT transgene could be regulated by GnRH, we treated ovariectomized females with antide, a GnRH antagonist. Antide suppressed transgene activity by 60%. Thus, the proximal promoter of the bovine LH beta subunit gene directs appropriate patterns of cell-specific expression and retains responsiveness to gonadal steroids and GnRH. In light of the robust activity of the LH beta promoter in transgenic mice, we suggest that this animal model can be exploited further to dissect the complex mechanisms that underlie gonadotrope-specific expression and hormonal regulation of the LH beta gene.

Animals↗
PubMed1994

Ifosfamide clinical pharmacokinetics.

This article reviews the metabolism and pharmacokinetics of ifosfamide and their implications for the cytostatic efficacy and toxicity pattern of this alkylating agent. Ifosfamide is a prodrug that requires biotransformation to become cytotoxic. It is a structural isomer of cyclophosphamide from which it differs only in having the chlorethyl functions on different nitrogen atoms. This causes a considerable change in initial metabolism, although overall metabolism remains the same. Beside the formation of 4-hydroxy-ifosfamide ('activated ifosfamide'), a second pathway with liberation of chloroacetaldehyde exists. Therefore, less activated drug is formed than during cyclophosphamide metabolism. This fact may well explain why higher doses of ifosfamide are required during treatment. Chloroacetaldehyde may account for the adverse effects and therapeutic effects of the parent drug. This metabolite has been associated with central nervous system toxicity during ifosfamide treatment and was shown to deplete intracellular glutathione concentrations. Glutathione depletion may support the activity of alkylating metabolites in tumour cells, thus overcoming the relative resistance of the cells to alkylating agents. Possibly, this mechanism explains the lack of complete cross-resistance between ifosfamide and cyclophosphamide as well as the greater antitumour activity of ifosfamide in some tumours. Urotoxicity of ifosfamide, which was the dose-limiting adverse effect, can be successfully attenuated by the use of mesna. Distinct pharmacokinetic properties of mesna are responsible for the fact that in contrast to other sulphydryl compounds the uroprotective activity of mesna does not imply a loss of therapeutic efficacy.

Administration, Oral↗
PubMed1994

Characterization of bile ducts in primary biliary cirrhosis and other liver diseases by immunohistochemical demonstration of Lewis(a) or b antigen.

We investigated Lewis(a) and Lewis(b) expression of bile ducts in 68 specimens from various kinds of liver disease. In addition, the number of IgM and IgG synthesizing plasma cells in the hepatic inflammatory reactions were immunostained and counted. We found a statistically significant decrease in the number of bile ducts in PBC (primary biliary cirrhosis) in comparison with either chronic active or persistent hepatitis (CAH/CPH). Bile ducts could be detected easily and constantly by their Lewis antigen expression. Isolated bile duct epithelial cells not apparent in H&E sections could be identified by Lewis(a) and b immunostaining. The number of plasma cells in PBC was significantly different than in (CAH/CPH). A large number of IgM plasma cells was a characteristic feature of PBC. However, neither counting of Lewis(a) and b positive bile ducts nor counting of IgM plasma cells was of definite diagnostic significance in the individual clinical case, since no cut-off value could be determined above or below which a PBC was ruled out or proven.

Bile Ducts↗
PubMed1993

Effects of temperature on the therapeutic efficacy and pharmacokinetics of ifosfamide.

The influence of tumor temperature (28, 32, 37, 39, 41, or 43 degrees C for 1 h) on the therapeutic efficacy of i.v. single bolus injections of ifosfamide (IFO) (32, 65, 125, or 250 mg/kg body weight) in human tumor xenografts (MX1 breast carcinoma) grown in nude mice (n = 240) was studied. Tumor temperature was controlled by water bath immersion. Sixty days after treatment the percentage of tumor-free survival was determined. For example, at 37 degrees C IFO in a dose of 65 mg/kg body weight led to 10% tumor-free survival in the treated animals. At 43 degrees C the same dose resulted in 60% tumor-free survival. A clear drug dose- and temperature-dependent increase of the therapeutic efficacy of an active oxazaphosphorine compound was also demonstrated in vitro. The concentrations of IFO and of 4-hydroxyifosfamide in blood and tumors at different body temperatures (controlled by water bath immersion) were determined over 120 min and WBC counts were obtained. The half-lives and the areas under the curve for IFO in blood were not significantly different at 37 degrees C and 41 degrees C. Since the half-life of IFO depends mainly on hepatic metabolism, the similarity of half-lives and of areas under the curve for IFO at 37 degrees C and 41 degrees C indicates a constant activation rate. However, significantly lower plasma concentrations of the activated drug at a liver (body) temperature of 41 degrees C, compared with 37 degrees C, were found, indicating a higher elimination rate. The concentration of the activated drug in the tumors within the initial 60 min at 41 degrees C, however, exceeded by > 2-fold that at 37 degrees C. The bone marrow toxicity of the same drug dose did not significantly increase with body temperature.

Animals↗
PubMed1993

Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis.

Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45-RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU-GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst-forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

Antibodies, Monoclonal↗
PubMed1993

Comparative pharmacokinetics of ifosfamide, 4-hydroxyifosfamide, chloroacetaldehyde, and 2- and 3-dechloroethylifosfamide in patients on fractionated intravenous ifosfamide therapy.

The initial metabolism of the oxazaphosphorine cytostatic ifosfamide (IF) consists of two different pathways: ring oxidation at carbon-4 forms the cytostatically active metabolite 4-hydroxyifosfamide (4-OH-IF, "activated ifosfamide"), whereas side-chain oxidation with liberation of the presumably neurotoxic compound chloroacetaldehyde (CAA) that may also be responsible for IF-associated nephrotoxicity results in the formation of the cytostatically inactive metabolites 2-dechloroethylifosfamide (2-DCE-IF) and 3-dechloroethylifosfamide (3-DCE-IF). The pharmacokinetics of IF and its metabolites were investigated in 11 patients with bronchogenic carcinoma receiving IF on a 5-day divided-dose schedule (1.5 g/m2 daily). Blood samples were drawn on days 1 and 5 for up to 24 h after the start of the IF infusion. IF, 2-DCE-IF, and 3-DCE-IF were simultaneously quantified by gas chromatography (GC) with an NIP flame-ionization detector (NPFID), CAA was determined by GC with an electron-capture detector (ECD), and the highly unstable compound 4-OH-IF was measured using a high-performance liquid chromatography (HPLC) assay with fluorometric detection of 7-OH-quinoline, which is formed by the condensation of 4-OH-IF-derived acrolein with m-aminophenol. As compared with the values obtained on day 1, on day 5 the terminal half-life and AUC values determined for IF were reduced by 30% (6.36 vs 4.06 h and 1781 vs 1204 nmol h ml-1, respectively), whereas the maximal concentration (Cmax) values were not affected significantly (199.1 vs 181.1 nmol ml-1). This known phenomenon is explained by autoinduction of hepatic IF metabolism and was paralleled by increased metabolite levels. The mean Cmax values determined for 4-OH-IF, CAA, 3-DCE-IF, and 2-DCE-IF (on day 1/on day 5) were 1.51/2.59, 2.69/4.85, 12.9/26.5, and 8.6/16.7 nmol ml-1, respectively. The corresponding AUC values were 11.3/16.5, 30.3/34.3, 146/354, and 111/209 nmol h ml-1, respectively. As calculated by intraindividual comparison, the mean Cmax (day 5): Cmax (day 1) ratios for 4-OH-IF, CAA, 3-DCE-IF, and 2-DCE-IF were 1.94*, 2.05*, 2.52*, and 2.33*, respectively; the corresponding AUC (day 5): AUC (day 1) ratios were 1.51*, 1.29, 2.34*, and 2.23*, respectively (* P < 0.05). These data reveal that during fractionated-dose IF therapy the cancerotoxic effect of the drug increases. If the assumed role of CAA in IF-associated neurotoxicity and nephrotoxicity is a dose-dependent phenomenon, the probability of developing these side effects would also increase during prolonged IF application.

Acetaldehyde↗
PubMed1993

[Resorbable implant materials in retinal detachment surgery. Initial animal experiment studies].

In detachment surgery after a mechanically stabile chorioretinal scar is at present full scleral indentation no longer mandatory. Following these considerations absorbable scleral implants have been developed and tested in an animal model. Cylindrical pieces of absorbable composite-material with a diameter of 5 mm were sutured as radial scleral explants in 18 rabbits. The composite material consisted of a polyglactin-polydioxanon ratio 7:1. The absorption time for polyglactin is 60 days, for polydioxanon 180 days. The explants produced initially in ultrasonographically measured buckle height between 3.2 and 4.0 mm. The clinical and ultrasonographical follow up study demonstrated a continuous decrease of buckle height. Two weeks after implantation there was a medium height of 2.0 mm after five weeks a medium elevation of 0.5 mm was found. Histopathological examinations showed incidence of a slight resorptive inflammation in the area of the buckle which was not evident after 12 weeks. No scleral infiltration or scleral thinning was found. The promising results have justified controlled clinical trials with the absorbable material which might allow to combine the advantages of silicone sponge explants (precise localization) with those of scleral buckles by inflatible balloons (reversibility).

Absorption↗
PubMed1993

[S-phase fraction and proportion of cells with high DNA content as prognostic factors of lymph node negative breast cancer].

Breast cancer is the most frequent female cancer in Western Europe. Roughly half of the patients have no axillary lymph node involvement at time of operation. In this group of patients adjuvant treatment should only be undertaken in the subgroup with a high risk of recurrence. Suitable significant prognostic parameters are needed to identify patients at risk. S-phase fraction and the percentage of cells with a high DNA content are two prognostic factors that are determined by measuring total nuclear DNA and cell cycle analysis. Both were analyzed in large studies and have shown their impact on prognosis. However, divergent results underline the necessity for larger studies with a longer follow-up.

Aneuploidy↗
PubMed1993

[Sensitivity of immunoscintigraphy in malignant melanoma of the choroid].

Between June, 1988 and May, 1991 30 patients with clinically suspected malignant melanoma were examined in succession by means of immunoscintigraphy and the 225.28 S antibody (tagged with Tc-99m). Retrospectively, the results showed a sensitivity of over 70% by SPECT examination. Positive accumulation of the antibody within the suspected melanoma gives reason to believe that this degree of accuracy can be improved by comparative application of unspecific, radioactively tagged proteins. In the absence of accumulation within clinically, and in particular echographically, confirmed malignant melanoma treatment should be performed.

Adult↗
PubMed1992

Attenuation of GTPase activity of recombinant G(o) alpha by peptides representing sequence permutations of mastoparan.

There is convincing evidence that the cytoplasmic domains of multispanning receptors interact with guanine nucleotide-binding proteins (G proteins). What are the rules governing these interactions? In an attempt to answer this question, we focused our attention on mastoparan, an amphiphilic tetradecapeptide from wasp venom, and on nine of its variants, produced by sequence permutation, which have altered amphiphilicity or no amphiphilicity at all. Mastoparan enhances the GTPase activity of recombinant G(o) alpha 5-fold in phospholipid vesicles. Like mastoparan, four of the synthetic variants can form amphiphilic alpha-helices and two of them indeed stimulate the GTPase activity of the G protein, whereas the other two have no effect. This confirms that the activation of certain G proteins by a number of peptides is mainly due to their cationic amphiphilicity. However, this structural feature is clearly not sufficient. The relative orientation of the positively charged residues as well as that of the hydrophobic side chains appear to be of fundamental importance. The other five peptides are not amphiphilic and do not enhance the rate of GTP hydrolysis. Surprisingly, three of them almost completely inhibit the G protein's intrinsic GTPase activity. This finding is of interest because of the possible role differential regulation of G protein activity can play in cellular functions.

Amino Acid Sequence↗
PubMed1992

Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).

Animals↗
PubMed1992

Identification of UAS elements and binding proteins necessary for derepression of Saccharomyces cerevisiae fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase is a key enzyme in gluconeogenesis and the FBP1 gene is not transcribed during growth with glucose. Genetic analysis indicated a positive regulation of FBP1 expression after exhaustion of glucose. By linker-deletion analysis, two upstream activation sites (UAS1 and UAS2) were localized and the respective UAS-binding factors (DAP I and DAP II for derepression activating protein) were identified by gel retardation. UAS1 and UAS2 span about 30 bp each, and are separated by approximately 30 bp. Both UAS sites act synergistically. Although UAS1 showed some similarities to the DNA-binding consensus for the general yeast activator Rap1, competition experiments and DEAE-chromatography proved that DAP I and Rap1 correspond to different proteins. Gel retardation by DAP I depended on carbon sources and did not occur in cells growing logarithmically with glucose, whereas a strong retardation signal was obtained with ethanol-grown cells. The present results suggest that DAP I and DAP II are the final regulatory elements for glucose derepression.

Base Sequence↗