Biomedical subjects

K Felix

Publications and source records attributed to K Felix.

At least 19 recordsLinked to original sources

PubMed2006

Hsulf-1 regulates growth and invasion of pancreatic cancer cells.

BACKGROUND: Hsulf-1 is a newly identified enzyme with arylsulphatase activity that can regulate the sulphation state of cell-surface heparan sulphate proteoglycans (HSPGs). In vitro overexpression of this enzyme in pancreatic cancer cells decreases responsiveness to fibroblastic growth factor-2, as Hsulf-1 is up regulated in primary pancreatic adenocarcinoma. AIM: To further analyse the functions of the Hsulf-1 enzyme in vitro and in vivo with respect to growth, invasion and tumorigenicity. METHODS AND RESULTS: Transfection of Panc-1 pancreatic cancer cells with a full-length Hsulf-1 expression vector resulted in increased invasiveness and adhesiveness. An in vivo xenograft nude mouse tumour model showed a markedly reduced growth potential of Hsulf-1-expressing Panc-1 cells, which correlated with a considerably lower proliferation rate. Hsulf-1-positive nude mouse tumours showed better development of interstitial matrix structures, with increased blood vessel density in these tumours. In an orthotopic model, Hsulf-1-positive tumours exhibited enhanced local invasiveness. In human primary pancreatic cancers there was strong staining for sulphated HSPGs, which was markedly reduced in metastatic tissue samples. CONCLUSION: Hsulf-1-mediated desulphation of HSPGs reduces the growth ability of Panc-1 pancreatic cancer cells, but increases the basal invasiveness of these cells, suggesting an important role of this enzyme in pancreatic cancer progression.

Adenocarcinoma↗

PubMed2005

Vanilloids in pancreatic cancer: potential for chemotherapy and pain management.

BACKGROUND: Success of chemotherapy and alleviation of pain are frequently less than optimal in pancreatic cancer patients, leading to increasing interest in new pharmacological substances, such as vanilloids. Our study addressed the question of whether vanilloids influence pancreatic cancer cell growth, and if vanilloids could be used for pain treatment via the vanilloid 1 receptor (VR1) in pancreatic cancer patients. METHODS: In vitro, the effect of resiniferatoxin (vanilloid analogue) on apoptosis and cell growth in pancreatic cancer cells--either alone, combined with 5-fluorouracil (5-FU), or combined with gemcitabine--was determined by annexin V staining, FACS analysis, and MTT assay, respectively. VR1 expression was evaluated on RNA and protein level by quantitative polymerase chain reaction and immunohistochemistry in human pancreatic cancer and chronic pancreatitis. Patient characteristics--especially pain levels--were registered in a prospective database and correlated with VR1 expression. RESULTS: Resiniferatoxin induced apoptosis by targeting mitochondrial respiration and decreased cell growth in pancreatic cancer cells without showing synergistic effects with 5-FU or gemcitabine. Expression of VR1 was significantly upregulated in human pancreatic cancer and chronic pancreatitis. VR1 expression was related to the intensity of pain reported by cancer patients but not to the intensity of pain reported by patients with chronic pancreatitis. CONCLUSIONS: Resiniferatoxin induced apoptosis in pancreatic cancer cells indicates that vanilloids may be useful in the treatment of human pancreatic cancer. Furthermore, vanilloid might be a novel and effective treatment option for neurogenic pain in patients with pancreatic cancer.

Adult↗

PubMed2004

Altered cytokine expression in tissues of mice subjected to simulated microgravity.

Space flight is known to induce microgravity-associated immune dysfunction in humans, non-human primates and rodents. To understand the mechanism underlying these defects, several studies in rodents have been conducted in a ground-based antiorthostatic suspension (AOS) model that would mimic the effects of microgravity. In all these in vivo studies that showed the effects on cytokine profiles actually investigated the ex vivo production from culturing the cells isolated from whole organism that was exposed to space flight and/or microgravity. So, the purpose of the study was to examine the in vivo expression of cytokines in mice in immunologically important tissue environments of mice that were subjected to AOS. Cytokines such as Interleukin-1beta, (IL-1beta), IL-2, IL-3, IL-6, Interferon-gamma (IFN-gamma) and Tumor Necrosis Factor-alpha (TNF-alpha) were measured by Enzyme Linked Immunosorbent Assay (ELISA) in the homogenates of spleen tissue, lymph nodes and also in serum of AOS mice and compared with that of control mice. AOS induced no change in the IL-3 levels, but IL-1beta was increased significantly whereas IL-2 levels decreased in spleen, lymph nodes and serum. IL-6 levels did not differ in spleen but were significantly increased in lymph nodes and serum of AOS mice. IFN-gamma levels in spleen did not change but showed nonsignificant reduction in lymph nodes and significant reduction in serum in response to AOS. TNF-alpha levels in spleen and serum were unchanged and increased in lymph nodes. This in vivo cytokine study confirms the earlier findings that microgravity-simulated conditions induce tissue-specific immune response.

Animals↗

PubMed2001

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

Animals↗

PubMed1999

Elevated mutant frequencies in lymphoid tissues persist throughout plasmacytoma development in BALB/c.lambdaLIZ mice.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, the mean mutant frequencies in the target gene, lacI, were found to be significantly increased in lymphoid tissues of congenic BALB/c.lambdaLIZ N5 mice in the terminal stage of a plasmacytoma induction experiment, 213-280 days after the first i.p. injection of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane). In plasmacytoma-bearing mice (n = 7), mutant frequencies in the spleens and mesenteric lymph nodes were elevated 2.46-fold and 5.35-fold, respectively, when compared with age-matched controls. In plasmacytoma-negative mice (n = 11), mutant frequencies were increased 2.30-fold (spleens) and 3.48-fold (mesenteric nodes). These results, interpreted in conjunction with our previous findings (K. Felix et al., Cancer Res., 58: 1616-1619, 1998) of approximately 3-fold elevations in pristane-induced splenic mutagenesis on day 42 postpristane, indicate that increased mutant levels in lymphoid tissues persist throughout plasmacytomagenesis in genetically susceptible BALB/c mice.

Animals↗

PubMed1999

Transgenic shuttle vector assays for assessing oxidative B-cell mutagenesis in vivo.

The recent development of transgenic mutagenicity assays provides new opportunities for evaluating mutagenic processes in vivo. To asses mutant frequencies in tissue B cells, we decided to take advantage of two such assays that utilize the transgenic shuttle vectors, lambda LIZ and pUR288. Our main interest in this research is to test two basic premises of inflammation-induced plasmacytoma development in genetically susceptible BALB/c mice; i.e., the possibility that plasmacytoma precursor cells may become targets of phagocyte-mediated oxidative mutagenesis in situ and the prospect that plasmacytoma susceptibility/resistance genes may contribute to these phenotypes by enhancing/reducing oxidative mutagenesis in B cells. Based on our preliminary experience with the lambda LIZ and pUR288 transgenic in vivo mutagenicity tests, we propose to employ these assays as broadly applicable tools for assessing overall mutagenesis during normal and aberrant (malignant) B-cell development. Furthermore, transgenic shuttle vector assays appear to lend themselves as ideal methods to associate general B-cell mutagenesis with the peculiar, B cell-typical somatic hypermutation processes that target the V(D)J gene segment, the proto-oncogene bcl-6 and perhaps other, still unknown loci.

Animals↗

PubMed1998

Association of elevated mutagenesis in the spleen with genetic susceptibility to induced plasmacytoma development in mice.

Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells.

Animals↗

PubMed1998

Tetravinyl-tetramethylcyclo-tetrasiloxane (tetravinyl D4) is a mutagen in Rat2lambda lacI fibroblasts.

Small fragments of silicone gels injected intraperitoneally have been used to induce plasmacytomas in genetically susceptible mice. Silicone oils, in contrast to silicone gels, are apparently not tumorigenic in the mouse plasmacytoma system. The reason for this difference as well as the mechanism of silicone gel-induced plasmacytoma development is poorly understood. We chose to examine the possibility that low molecular wt silicone compounds such as siloxanes, leaking from the complex silicone gel matrix into the surrounding tissue, may be mutagenic. We postulate that this mutagenicity may be a critical determinant of the plasmacytoma inducing potency of silicone gels. Six siloxane compounds, either linear or cyclic di-, tri-, or tetrasiloxanes substituted with methyl or vinyl moieties, were selected as model compounds to study mutagenicity in Rat2lambda lacI fibroblasts in vitro. Using phage lambda-derived lacI/lacZ genes as target/reporter genes to quantitate mutagenesis, and gamma-cyclodextrin as vehicle to effectively deliver siloxanes, we found that exposure to 50 microM of tetravinyl-tetramethylcyclo-tetrasiloxane (tetravinyl D4) resulted in a modest 1.7-fold increase of mutant frequencies over controls in Rat2lambda lacI cells. In related toxicity experiments, tetravinyl D4 was shown to perturb lipid membranes leading to a loss of cytosolic glutathione (GSH), which by itself resulted in a 1.5-fold increased mutant rate in Rat2lambda lacI cells. We conclude that certain siloxanes may act as direct mutagens in mammalian cells. In addition, siloxane-induced mutagenicity may be enhanced by the depletion of intracellular GSH caused by the interaction of lipophilic siloxanes with cell membranes.

Animals↗

PubMed1997

In vitro mutagenicity of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane).

Pristane is known to induce a distinct type of B-cell-derived malignant lymphoma, plasmacytoma, after administration into the peritoneal cavity of genetically susceptible BALB/cAnPt mice. Since the mechanism of pristane-induced plasmacytoma development is poorly understood, we chose to examine the possibility that pristane is mutagenic in rodent cells and decided to use bacteriophage lambda-derived lacI/lacZ genes as target/reporter to quantitate mutagenesis. Here we show that in vitro exposure to micromolar amounts of pristane, delivered as an inclusion complex with beta-cyclodextrin, resulted in 1.7-fold and 6.2-fold increases of mutant frequencies over controls in a cell line of rat fibroblasts and primary mouse B lymphocytes, respectively. We conclude that pristane can be mutagenic to mammalian cells, yet are currently unable to explain the mechanism of mutagenicity. It is suggested that B-cell mutagenesis contributes to the plasmacytomagenic activity of pristane in vivo.

Amino Acid Sequence↗

PubMed1997

Elevated mutant frequencies in gene lacI in splenic lipopolysaccharide blasts after exposure to activated phagocytes in vitro.

The interaction of B lymphocytes with phagocytes is critical for shaping the humoral immune response, as well as various aspects of normal and malignant B cell development, and has therefore been studied by immunologists in great detail. However, one potential outcome of this confrontation is often neglected, namely the mutagenicity of phagocytes to B lymphocytes. We are interested in phagocyte-induced B cell mutagenesis and have conducted a feasibility study on the utility of a transgenic reporter assay to evaluate mutant frequencies in B cells that have encountered phagocytes. An in vitro co-incubation system was designed in which splenic lipopolysaccharide (LPS) blasts carrying a phage lambda-derived lacI transgene were exposed to pristane-elicited peritoneal exudate cells (PEC). Mutant frequencies in LPS blasts were significantly increased (up to 6-fold) when the cells were co-incubated with PEC that had been stimulated by phorbol myristate acetate to undergo an oxidative burst. The lacI-based transgenic mutation assay proved also useful for assessing mutagenicity in vivo, as demonstrated by the detection of elevated mutant frequencies in the spleen (3-fold) and the inflammatory granuloma (4.7-fold) obtained from pristane-treated mice. We propose to utilize the lacI-based transgenic mutagenesis assay as a tool to evaluate mutational levels during normal and aberrant B cell differentiation.

Animals↗

PubMed1996

Cytotoxicity and membrane damage in vitro by inclusion complexes between gamma-cyclodextrin and siloxanes.

Inclusion complexes of gamma-cyclodextrin and octamethylcyclotetrasiloxane (D4), decamethyltetrasiloxane (M10TS), and 1,3,5,7-tetramethyltetravinylcyclotetra - siloxane (TMTV-D4) were prepared to compare the cytotoxic effects of siloxanes in vitro. In these preparations, the hydrophobic siloxanes are surrounded by a hydrophilic shell of eight circularly linked D-glucose molecules (gamma-cyclodextrin), and upon contact with plasma membranes the siloxane molecule can intercalate into the lipid bilayer of the cell membrane. XRPC24, 2-11 plasmacytoma, CH12.LX lymphoma and P388D1 macrophage-like cells were used as indicator cells in toxicity assays. Using an MTT tetrazolium reduction to formazan test, a colorimetric method to determine the number of viable cells, the 50% minimal lethal doses (CD50) for the siloxane compounds were found to range from 30 to 50 microM. Sublethal doses (e.g., 15 microM and lower) resulted in the loss of lactate dehydrogenase (LDH) and glutathione (GSH) from the cytosolic compartment of the target cells and thus indicated cytotoxicity. Treatment of macrophages with siloxanes resulted in a higher production of interleukin-6 (IL-6) than was exhibited by untreated macrophages. The B9 cell bioassay of these treated cells showed as much as a 10 fold higher production (500 U/ml) of IL-6 than did the untreated cells. The degree of increase was dependent on the compound and concentration used. The results of this study show that low molecular weight siloxanes produce lethal effects on B-lymphocyte derived target cells in vitro and permeabilize the plasma membranes at lower sublethal concentrations.

Animals↗

PubMed1996

The antioxidative activity of the mucoregulatory agents: ambroxol, bromhexine and N-acetyl-L-cysteine. A pulse radiolysis study.

Ambroxol and bromhexine are shown to be scavengers of both superoxide and hydroxyl radicals as determined by pulse radiolysis experiments. The dismutation of superoxide was accelerated 3-fold by bromhexine and 2.5-fold by ambroxol over the rate of spontaneous dismutation. The reaction constants of hydroxyl radicals with bromhexine and ambroxol were determined by competition kinetics to be 1.58 +/- 0.15 x 10(10) M-1S-1 and 1.04 +/- 0.1 x 10(10) M-1S-1, respectively. N-acetyl-L-cysteine also reacted with hydroxyl radicals (1.28 +/- 0.14 x 10(10) M-1S-1) but not with superoxide radical. These effects may be clinically relevant in the treatment of oxidant-associated lung damage induced by inflammatory agents and/or environmental pollutants.

Acetylcysteine↗

PubMed1996

Allergens of horse dander: comparison among breeds and individual animals by immunoblotting.

Some patients who are allergic to horses have reported that they can tolerate certain breeds, and the presence of breed-specific allergens has been suggested. Breeders and patients with asthma have claimed that Bashkir horses are nonallergenic. We used sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting to determine IgE-binding profiles of extracts of dander obtained from horses of several breeds. We found considerable inter-breed and within-breed variation but no breed-specific allergens. Danders from all breeds investigated contained the most important allergens, and the allergenic content of dander from Bashkir horses was similar to that of other breeds. Difference in scale production could account for differences in sensitivity to breeds and individual horses.

Allergens↗

PubMed1995

Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats.

The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.

Aging↗

PubMed1995

Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium.

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.

Animals↗

PubMed1994

Induction of oxidative stress and protection against hydrogen peroxide-mediated cytotoxicity by the superoxide dismutase-mimetic complex copper-putrescine-pyridine.

The low molecular weight Cu2Zn2-superoxide dismutase (SOD) active centre analogue copper-putrescine-pyridine (Cu-PuPy, N,N'-bis(2-pyridylmethylene)1,4-butanediamine(N,N',N",N")-Cu (II)- -diperchlorate) has been shown to dismutate superoxide with high efficiency. In the presence of glutathione it sustains the production of H2O2 via redox cycling. We investigated the influence of Cu-PuPy on the glutathione status and the clonogenic survival of Chinese hamster ovary (CHO) cells. At 0.05 mM Cu-PuPY was not toxic and exerted only a minor effect on cellular glutathione. At Cu-PuPy concentrations of 0.1-0.5 mM glutathione became increasingly oxidized and was depleted during treatment while toxicity dramatically increased. The time course of toxicity was unusual: after passing a minimum at 50 or 100 min (0.5 mM or 0.2 mM Cu-PuPy, respectively), clonogenic survival increased by two orders of magnitude in the following 50 min. On the other hand, Cu-PuPy protected cells effectively against toxic doses of hydrogen peroxide. We conclude that Cu-PuPy combines a prooxidant and an antioxidant mode of action that sequentially modify the survival response of CHO cells: initial production of hydrogen peroxide by Cu-PuPy-catalysed glutathione oxidation leads to the intracellular accumulation of potentially toxic radical intermediates that may be inactivated via superoxide dismutation upon further treatment with Cu-PuPy.

Animals↗

PubMed1993

A pulse radiolytic study on the reaction of hydroxyl and superoxide radicals with yeast Cu(I)-thionein.

In a pulse radiolytic study employing aqueous intact yeast copper(I)-thionein at pH 7 it was shown that both superoxide and hydroxyl radicals efficiently react with this Cu(I)- and thiolate-rich protein. The reaction constant of hydroxyl radicals with Cu(I)-thionein was determined by competition kinetics and was 2.2 x 10(11) M-1 s-1 at a rate close to a diffusion-controlled limit. The reaction of Cu(I)-thionein with superoxide was also successful and proceeded at a rate of 7.5 x 10(6) M-1 s-1. According to chiroptical and luminescence emission measurements minor oxidation of the copper(I)-thiolate oligonuclear binding centres was observed, leading to the release of some Cu(II). It is important to realise the dual reactivity of this yeast Cu(I)-thiolate protein in controlling copper transport and storage as well as its distinct role in the scavenging of free radicals.

Binding Sites↗

PubMed1993

Intestinal administration of copper and its transient release into venous rat blood serum concomitantly with metallothionein.

The molecular side of copper transport in biological systems is unknown. It was attempted to examine the copper and metallothionein (MT) release into the portal blood in rats in vivo. After direct administration of Cu(II) into the jejunum the copper and MT levels were distinctively higher in the portal venous serum compared with that of the vena cava inferior. MT in gel filtrated serum samples was analyzed immunologically employing ELISA and a monoclonal antibody to rat MT-I. Affinity chromatography on Protein A-Sepharose resulted in a higher immunoreactivity in the portal compartment as deduced from an elevated MT-antibody complex. It is assumed that MT serves as a genuine transport system for cuprous copper during the mucosal-to-serosal flux of this biologically important transition metal.

Animals↗