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K R Huber

Publications and source records attributed to K R Huber.

11 recordsLinked to original sources
PubMed1998

Restriction digest PCR (RD-PCR) for the analysis of gene mutations. Application to Ki-ras.

The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, "enrichment PCR" must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5'primer (...GGA CCT GGT...) for an enzyme (BstNI) (5'CC!WGG 3') to differentiate between wild-type sequence (...GGA GCT GGT...) (during amplification, the G is replaced by a C) and mutated sequences (_...GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the second round altogether. The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.

Base Sequence↗
PubMed1997

The N-methyl-D-aspartate (NMDA) receptor glycine site antagonist ACEA 1021 does not produce pathological changes in rat brain.

ACEA 1021 is a potent, selective N-methyl-D-aspartate (NMDA) receptor glycine site antagonist under clinical evaluation as a neuroprotectant for stroke and head trauma. The potential of ACEA 1021 to produce morphologic changes in cerebrocortical neurons of the rat was assessed since it is known that noncompetitive (e.g., MK-801) and competitive (e.g., CGS 19755)NMDA receptor antagonists produce neuronal vacuolization and necrosis in the rat posterior cingulate/retrosplenial cortex. Male and female adult rats were treated intravenously with either vehicle (Tris) or 10 mg/kg or 50 mg/kg ACEA 1021. MK-801 (5 mg/kg, s.c.) served as positive control. Whereas MK-801 produced characteristic neuronal vacuolization and necrosis in the posterior cingulate/retrosplenial cortex, neither dose of ACEA 1021 had any effect on neuronal morphology. The absence of neuropathological changes in rats supports the further clinical evaluation of ACEA 1021 for stroke and head trauma, and suggests that glycine site antagonists may be devoid of neurotoxic potential.

Animals↗
PubMed1996

Detection of common hepatitis C virus subtypes with a third-generation enzyme immunoassay.

The causal agent of most posttransfusion non-A and non-B hepatitis infections was characterized in 1989 by molecular biological techniques as a positive-stranded, enveloped RNA virus, designated hepatitis C virus (HCV). Only since 1990 has it been possible to screen for an infection with antibody tests or direct amplification assays for the nucleic acid (i.e., reverse-transcription polymerase chain reaction [PCR]). However, these nucleic acid based tests are time consuming and rather expensive. Recently, third-generation enzyme immunoassays (EIAs) for HCV infection were introduced (i.e., Abbott Laboratories, North Chicago, IL; Ortho Diagnostics, Inc., Raritan, NJ; and Roche Diagnostics, Basel, Switzerland). The Roche Diagnostics EIA has been evaluated with our patient population. To this end, 1,090 samples were assayed by both EIA and PCR; 946 of all samples (87%) were negative, and 107 samples (9.8%) were positive by both tests. Thirty of the patients (2.7%) showed antibodies but no detectable virus, whereas 7 of all patients tested (0.6%) were PCR-positive but had not yet developed antibodies to the virus. Of these 7 patients, only one showed normal serum transaminases. Virus strain subtyping and quantification of viral load on the positive samples in parallel have been performed, and the fact that the EIA detects all the virus subtypes found in our hospital can be inferred. No correlation between subtypes, viral load, and immune response could be measured with this antibody test. Our results indicate that, in most circumstances (except in settings where immunocompromised patients are abundant), this EIA can replace the much more expensive PCR tests for the routine screening for HCV infection.

Adolescent↗
PubMed1989

Human tumor cells resistant to verapamil.

The efficacy of the calcium channel blocker verapamil for enhancing at low concentrations the cytotoxicity of unrelated antineoplastic drugs and for inhibiting at high concentrations cell proliferation has stimulated interest in the underlying mechanisms of these two diverse effects. We have selected two human brain tumor cell lines (a TE671 medulloblastoma and a A172 glioma line) for resistance against 100 uM verapamil to aid in the elucidation of the mechanism of verapamil's antiproliferative effect. Our first experiments on the selected TE671 medulloblastoma cells show that, in the presence of 100 uM verapamil, these cells grow at a rate similar to that observed for the sensitive cells in the absence of verapamil. This resistant clone continues to exhibit resistance toward verapamil for at least three days after the verapamil has been removed from the growth medium. In contrast to the sensitive cells, the resistant cells show only slight cell cycle phase alterations after removal of verapamil from the growth medium. This, together with an unchanged c-myc gene expression after removal of verapamil, indicates a stable phenotypic alteration that is responsible for the exhibited resistance toward the antiproliferative effects of the drug. Experiments designed to elucidate the mechanism of resistance showed that these cells are not cross-resistant to the antineoplastic drugs vincristine and adriamycin. Also, the resistance is not accompanied by increased amounts of the 170-180 kDa P-glycoprotein that has been implicated in resistance phenomena of cancer cells towards antineoplastic drugs.

Cell Cycle↗
PubMed1989

Effect of verapamil on cell cycle transit and c-myc gene expression in normal and malignant murine cells.

Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines (Schmidt et al., Cancer Res., 48, 3617, 1988). We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.

Animals↗
PubMed1988

Antiproliferative effect of verapamil alone on brain tumor cells in vitro.

Recent studies have shown that the calcium channel blockers, when combined with standard anticancer drugs, help overcome resistance that often develops to those drugs. Little is known about the effects of the calcium channel blockers themselves on tumor cells. We have studied the effects of one calcium channel blocker, verapamil, on human tumor cell lines in vitro. Our results show a reversible, antiproliferative action of verapamil on human medulloblastoma, pinealoblastoma, glioma, and neuroblastoma tumor lines established from pediatric patients. Growth rates are inhibited 10 to 100% by 10 to 100 microM verapamil with 50% inhibition occurring between 25 and 50 microM verapamil. No cell line proliferates in 100 microM verapamil, yet washing the cells after 72 h of incubation with 100 microM verapamil results in resumed cell growth. Growth inhibition is accompanied by dose-dependent decreases in DNA, RNA, and protein synthesis which occur within minutes after addition of verapamil. DNA flow cytometry on propidium iodide-stained nuclei shows that, after incubation for 48 h with 100 microM verapamil, the medulloblastoma and neuroblastoma tumor lines as well as normal, human foreskin and lung fibroblast cell lines are reversibly blocked throughout the cell cycle with slight increases in G1. Verapamil appears to have no effect on nucleic acid precursors or on calcium influx or efflux in human medulloblastoma cells.

Calcium↗
PubMed1988

Uptake of glutamine antimetabolites 6-diazo-5-oxo-L-norleucine (DON) and acivicin in sensitive and resistant tumor cell lines.

The uptake system for 6-diazo-5-oxo-L-norleucine (DON) was studied in mouse P388 leukemia cells. The DON transport system was found to resemble that of another glutamine antimetabolite, Acivicin, in its strong temperature dependence, utilization of the "L" transport system, inhibition by glutamine but not by glutamate, potent inhibition by p-chloromercuribenzene sulfonate, Na+, and only minimal inhibition by various energy poisons. A Km of approximately 70 microM and a Vmax of 3.4 nmoles/10(6) cells/min was calculated for this cell line. The accumulated DON was not metabolized by P388 cells and moderate efflux occurred at 37 degrees C. The DON transport characteristics of a DON-resistant P388 cell line (100 times ID50 of parent line) were similar to those of the DON-sensitive parent line, indicating that altered drug transport may not be involved in development of resistance to this antimetabolite. The finding that an Acivicin-resistant subline of P388 cells which exhibited good transport of DON showed negligible transport of Acivicin suggests different modes of resistance towards the two glutamine antimetabolites.

Animals↗
PubMed1987

Superoxide dismutase-like activities of copper(II) complexes tested in serum.

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.

Animals↗