Biomedical subjects

L Madsen

Publications and source records attributed to L Madsen.

At least 19 recordsLinked to original sources

PubMed2001

Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status.

During aerobic metabolism, a small amount of partially reduced oxygen is produced, yielding reactive oxygen species (ROS). Peroxisomes and mitochondria are major contributors to cellular ROS production, which is normally balanced by consumption by antioxidants. The fatty acid analogue tetradecylthioacetic acid (TTA) promotes mitochondrial and peroxisomal proliferation, and may induce oxidative stress and change the growth potential of cancer cells. In the present study, we found that TTA reduced [(3)H]thymidine incorporation in the glioma cell lines BT4Cn (rat), D54Mg (human), and GaMg (human) in a dose- and time-dependent manner. The 50% inhibitory TTA doses were approximately 125 microM for BT4Cn and D54Mg cells and 40 microM for GaMg cells after 4 days. alpha-Tochopherol counteracted this inhibition in GaMg cells. TTA enhanced the oxidation of [1-(14)C]palmitic acid, which could be explained by stimulation of enzymes involved in peroxisomal (fatty acyl-CoA oxidase) and/or mitochondrial (carnitine palmitoyltransferase) fatty acid oxidation. The glutathione content and the activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase were differentially affected. Increased malondialdehyde (MDA) production was seen in TTA-treated GaMg and D54Mg cells, but not in BT4Cn cells, in vitro. In BT4Cn tumor tissue from TTA-treated rats, MDA was increased while the alpha-tocopherol content tended to decrease. TTA increased the level of cytosolic cytochrome c in BT4Cn cells, which suggests induction of apoptotic cascades. Although several mechanisms are likely to be involved in the TTA-mediated effects on growth, we propose that modulation of cellular redox conditions caused by changes in fatty acid metabolism may be of vital importance.

Animals↗

PubMed2001

Effects of long-chain monounsaturated and n-3 fatty acids on fatty acid oxidation and lipid composition in rats.

Long-chain n-3 fatty acids and fat fish are reported, among multiple physiological properties, to enhance peroxisomal beta-oxidation and effect triacylglycerol status. Long-chain n-3 and monounsaturated fatty acids are the main portion of fatty acids in fat fish. The individual effect of long-chain monounsaturated fatty acids on beta-oxidation and fatty acid composition was tested and compared to the effect of n-3 polyunsaturated and saturated fatty acids in a 3-week feeding experiment of rats. To explore the contribution from long-chain monounsaturated fatty acids in these aspects, the effect of long-chain n-3 and monounsaturated fatty acids on mitochondrial and peroxisomal beta-oxidation was compared, as well as fatty acid composition of adipose tissue, liver and serum. Fatty acid oxidase, palmitoyltransferase I and II activities, the amount of serum lipids, and the fatty acid composition of lipid fractions from the organs were analysed. The peroxisomal beta-oxidation was enhanced by the n-3 fatty acids, whereas a small, significant increase with the monounsaturated fatty acids was observed. There was a stimulation of the mitochondrial oxidation with the n-3 fatty acids, but monounsaturated fatty acids gave a small, nonsignificant decrease. With n-3 fatty acids there was a considerable decrease in the levels of serum triacylglycerol, phospholipids, free fatty acids and total cholesterol, while there were only minor effects of monounsaturated fatty acids. As judged from the fatty acid composition data, there was a mobilization on n-3 fatty acids from the adipose tissue to liver and plasma with the n-3 diet. This observation was also seen with the monounsaturated fatty acid-enriched diet. In conclusion, monounsaturated fatty acids seemed to stimulate peroxisomal beta-oxidation and to increase plasma triacylglycerol, whereas the mitochondrial oxidation was slightly decreased.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2001

Local tumor control with salvage cryotherapy for locally recurrent prostate cancer after external beam radiotherapy.

PURPOSE: We identified variables associated with a positive prostate biopsy after salvage cryotherapy in patients in whom initial external beam radiotherapy for prostate cancer failed to improve our cryotherapy technique, optimize local control and improve our patient selection criteria for salvage cryotherapy. MATERIALS AND METHODS: Between July 1992 and January 1995, 145 patients underwent salvage cryotherapy. Post-cryotherapy sextant prostate biopsies were performed in 107 cases. We evaluated certain variables on univariate and multivariate analysis as predictors of a positive biopsy after cryotherapy, including the type of previous therapy, tumor stage and grade at initial diagnosis, prostate volume, pre-cryotherapy prostate specific antigen (PSA), number of positive biopsy cores before cryotherapy, PSA nadir after cryotherapy, stage and grade of local recurrence, number of cryoprobes, number of freeze-thaw cycles and use of a urethral warming catheter during cryotherapy. RESULTS: Biopsies were positive in 23 cases (21%) after salvage cryotherapy. Variables associated with a positive biopsy on univariate analysis were initial stage, precryotherapy PSA, PSA nadir after cryotherapy, number of cryoprobes, number of freeze-thaw cycles and a history of chemotherapy (p = 0.005, 0.027, 0.001, 0.009, 0.018 and 0.008, respectively). Variables that remained associated with a positive biopsy on multivariate analysis were the number of probes used and post-cryotherapy PSA nadir (p = 0.013 and 0.019, respectively). CONCLUSIONS: Patients with initial clinical stage T1-2N0M0 disease and PSA no more than 10 ng./ml. have a higher rate of negative biopsies after salvage cryotherapy. Therefore, they are better candidates for salvage cryotherapy for locally recurrent prostate adenocarcinoma after external beam radiotherapy. To optimize the potential for local control the technique of salvage cryotherapy should include 2 freeze-thaw cycles and a minimum of 5 cryoprobes. Detectable PSA after salvage cryotherapy is a strong predictor of local failure.

Biopsy↗

PubMed2000

Characterisation of streptomycin resistance determinants in Danish isolates of Salmonella Typhimurium.

Fifty six Danish streptomycin (Sm) resistant isolates of Salmonella enterica serotype Typhimurium from pigs (n=34), calves (n=3) and humans (n=19) were characterised with respect to co-resistances (14 drugs), transferability of Sm-resistance by conjugation, genetic determinants encoding Sm-resistance and diversity with respect to localisation of genes in the genome and DNA-sequences. Forty-six strains carried resistance(s) other than Sm-resistance. Nineteen different co-resistance patterns were observed and tetracycline was the most commonly observed resistance in these patterns. In 22 of the strains, Sm-resistance was transferred by conjugation. Eleven strains contained the gene aadA only, six strains contained aadA+strA+strB, and 35 strains contained strA+strB. Partial sequences of aadA were obtained from four strains. Three strains showed identical sequences to a published aadA sequence from the transposon Tn7, and in one strain the sequence showed one synonymous substitution compared to this sequence. Partial sequences were obtained of strA and strB in seven strains. The sequence of strB was identical to the published sequence of the plasmid RSF1010 in all strains. All seven sequences of strA were identical and differed from the sequence of strA in RSF1010 by two non-synonymous substitutions.

Animals↗

PubMed2000

Peroxisome proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity.

Fibrates and glitazones are two classes of drugs currently used in the treatment of dyslipidemia and insulin resistance (IR), respectively. Whereas glitazones are insulin sensitizers acting via activation of the peroxisome proliferator-activated receptor (PPAR) gamma subtype, fibrates exert their lipid-lowering activity via PPARalpha. To determine whether PPARalpha activators also improve insulin sensitivity, we measured the capacity of three PPARalpha-selective agonists, fenofibrate, ciprofibrate, and the new compound GW9578, in two rodent models of high fat diet-induced (C57BL/6 mice) or genetic (obese Zucker rats) IR. At doses yielding serum concentrations shown to activate selectively PPARalpha, these compounds markedly lowered hyperinsulinemia and, when present, hyperglycemia in both animal models. This effect relied on the improvement of insulin action on glucose utilization, as indicated by a lower insulin peak in response to intraperitoneal glucose in ciprofibrate-treated IR obese Zucker rats. In addition, fenofibrate treatment prevented high fat diet-induced increase of body weight and adipose tissue mass without influencing caloric intake. The specificity for PPARalpha activation in vivo was demonstrated by marked alterations in the expression of PPARalpha target genes, whereas PPARgamma target gene mRNA levels did not change in treated animals. These results indicate that compounds with a selective PPARalpha activation profile reduce insulin resistance without having adverse effects on body weight and adipose tissue mass in animal models of IR.

Adipose Tissue↗

PubMed2000

Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids.

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.

Acyl-CoA Oxidase↗

PubMed2000

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification.

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.

Animals↗

PubMed2000

Detection and molecular mass determination of an HIV replication-enhancing female genital tract factor using a blot bioassay.

We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot technique that involves nonreducing SDS-PAGE of CVL samples and electrophoretic transfer onto nitrocellulose paper followed by incubation of paper slices with HIV-infected monocytoid U1 cells. The slices with HIF bioactivity were detected by increased HIV production and measured by an HIV core protein (p24) ELISA. We refer to this technique as the "BioBlot" assay. The BioBlot assay successfully detected bioactivity of HIF anchored onto nitrocellulose and determined that HIF has a molecular mass of about 14 kDa. Paper slices with HIF-negative CVL samples as well as nitrocellulose paper samples without CVL did not enhance HIV production. This finding suggested that SDS-PAGE and nitrocellulose binding do not functionally alter the bioactive domain(s) of HIF structure. In addition to the detection of HIF bioactivity anchored to nitrocellulose and HIF molecular mass determination, the BioBlot technique offers an alternative, rapid method for other applications. These include the study of receptor-ligand interactions of mucosal proteins, direct bioactivity testing and molecular mass determination of secretory substances.

Biological Assay↗

PubMed1999

Methylated eicosapentaenoic acid and tetradecylthioacetic acid: effects on fatty acid metabolism.

We introduced methyl or ethyl groups to the 2- or 3-position of the eicosapentaenoic acid (EPA) molecule to investigate whether the branching of EPA could influence its hypolipidemic effect in rats. The most effective branching involved two methyl groups in the 2-position and one methyl group in the 3-position. These EPA derivatives increased hepatic mitochondrial and peroxisomal beta-oxidation and decreased plasma lipids concomitant with suppressed acetyl-coenzyme A (CoA) carboxylase (EC 6.4.1.2) and fatty acid synthase (EC 2.3.1.85) activities. This was followed by elevated activities of camitine O-palmitoyltransferase (EC 2.3.1.21) and possibly 2,4-dienoyl-CoA reductase (EC 1.3.1.34), as well as induced mRNA levels of these enzymes and fatty acyl-CoA oxidase. The fatty acid composition in liver changed, with an increased 18:1 n-9 content, whereas the expression of delta9-desaturase remained unchanged. We investigated the flux of fatty acids in cultured hepatocytes, and found that oxidation of [1-14C]-labeled palmitic acid increased but the secretion of palmitic acid-labeled triglycerides decreased after addition of 2-methyl-EPA. The fatty acyl-CoA oxidase (EC 1.3.3.6) activity in these cells remained unchanged. A significant negative correlation was obtained between palmitic acid oxidation and palmitic acid-labeled synthesized triglycerides. To investigate whether the hypolipidemic effect occurred independently of induced peroxisomal beta-oxidation, we fed rats 2-methyl-tetradecylthioacetic acid. This compound increased the peroxisomal but not the mitochondrial beta-oxidation, and the plasma lipid levels were unchanged. In conclusion, EPA methylated in the 2- or 3-position renders it more potent as a hypolipidemic agent. Furthermore, this study supports the hypothesis that the mitochondrion is the primary site for the hypolipidemic effect.

Animals↗

PubMed1999

In contrast with docosahexaenoic acid, eicosapentaenoic acid and hypolipidaemic derivatives decrease hepatic synthesis and secretion of triacylglycerol by decreased diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation.

Hypolipidaemic fatty acid derivatives and polyunsaturated fatty acids decrease concentrations of plasma triacylglycerol by mechanisms that are not fully understood. Because poor susceptibility to beta- and/or omega-oxidation is apparently a determinant of the peroxisome proliferating and hypolipidaemic capacity of fatty acids and derivatives, the relative importance of activation of the peroxisome-proliferator-activated receptor alpha (PPARalpha), fatty acid oxidation and triacylglycerol synthesis were examined. We have compared the effects of differentially beta-oxidizable fatty acids on these parameters in primary cultures of rat hepatocytes. Tetradecylthioacetic acid (TTA), 2-methyleicosapentaenoic acid and 3-thia-octadecatetraenoic acid, which are non-beta-oxidizable fatty acid derivatives, were potent activators of a glucocorticoid receptor (GR)-PPARalpha chimaera. This activation was paradoxically reflected in an substantially increased oxidation of [1-(14)C]palmitic acid and/or oleic acid. The incorporation of [1-(14)C]palmitic acid and/or oleic acid into cell-associated and secreted triacylglycerol was decreased by 15-20% and 30% respectively with these non-beta-oxidizable fatty acid derivatives. The CoA ester of TTA inhibited the esterification of 1, 2-diacylglycerol in rat liver microsomes. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) activated GR-PPARalpha. EPA increased the oxidation of [1-(14)C]palmitic acid but DHA had no effect. The CoA ester of EPA inhibited the esterification of 1, 2-diacylglycerol, whereas DHA-CoA had no effect. The ratio between synthesized triacylglycerol and diacylglycerol was lower in hepatocytes cultured with EPA in the medium compared with DHA or oleic acid, indicating a decreased conversion of diacylglycerol to triacylglycerol. Indeed, the incorporation of [1-(14)C]oleic acid into secreted triacylglycerol was decreased by 20% in the presence of EPA. In conclusion, a decreased availability of fatty acids for triacylglycerol synthesis by increased mitochondrial beta-oxidation and decreased triacylglycerol formation caused by inhibition of diacylglycerol acyltransferase might explain the hypolipidaemic effect of TTA and EPA.

Acyltransferases↗

PubMed1999

Mice lacking all conventional MHC class II genes.

MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCII(Delta/Delta) mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aalpha and Abeta knockout animals, notably a dearth of CD4(+) lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCII(Delta/Delta) mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering "humanized" mouse models of human MHC-II-associated autoimmune disorders.

Animals↗

PubMed1999

Reproducible methods for experimental infection with Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss.

Experiments were done in order to achieve a reproducible method that can be used to infect rainbow trout Oncorhynchus mykiss with Flavobacterium psychrophilum, the causal agent of coldwater disease and rainbow trout fry syndrome. The main method investigated was intraperitoneal injection, and this method was tested using isolates with different elastin-degrading profiles and representing different serotypes. Injecting trout, average weight 1 g, with 10(4) CFU (colony-forming units) per fish caused cumulative mortalities around 60 to 70%. The virulent strains belonged to certain serotypes and degraded elastin. The intraperitoneal injection challenge method could be used on larger fish, but the infection dose was 10(7) CFU per fish before mortalities occurred. Bath infection and bath infection in combination with formalin treatment (stress) seemed to be reproducible methods that could be used as alternatives to the intraperitoneal method, although the mortalities among infected trout were lower. The results of investigated methods were influenced by parameters such as the challenge isolate, number of fish in the tank affecting the infection pressure, origin of fish and weight of fish.

Animals↗

PubMed1999

Beneficial effects of fibrates on apolipoprotein A-I metabolism occur independently of any peroxisome proliferative response.

BACKGROUND: In humans, fibrates are frequently used normolipidemic drugs. Fibrates act by regulating genes involved in lipoprotein metabolism via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in liver. In rodents, however, fibrates induce a peroxisome proliferation, leading to hepatomegaly and possibly hepatocarcinogenesis. Although this peroxisome proliferative response appears not to occur in humans, it remains controversial whether the beneficial effects of fibrates on lipoprotein metabolism can occur dissociated from such undesirable peroxisomal response. Here, we assessed the influence of fenofibrate on lipoprotein metabolism and peroxisome proliferation in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. METHODS AND RESULTS: First, we demonstrate that in normal rabbits, fenofibrate given at a high dose for 2 weeks does not influence serum concentrations or intestinal mRNA levels of the HDL apolipoprotein apoA-I. Therefore, the study was continued with human apoA-I transgenic rabbits that overexpress the human apoA-I gene under control of its homologous promoter, including its PPAR-response elements. In these animals, fenofibrate increases serum human apoA-I concentrations via an increased expression of the human apoA-I gene in liver. Interestingly, liver weight or mRNA levels and activity of fatty acyl-CoA oxidase, a rate-limiting and marker enzyme of peroxisomal beta-oxidation, remain unchanged after fenofibrate. CONCLUSIONS: Expression of the human apoA-I transgene in rabbit liver suffices to confer fibrate-mediated induction of serum apoA-I. Furthermore, these data provide in vivo evidence that the beneficial effects of fibrates on lipoprotein metabolism occur mechanistically dissociated from any deleterious activity on peroxisome proliferation and possibly hepatocarcinogenesis.

Acyl-CoA Oxidase↗

PubMed1999

Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase and carnitine palmitoyltransferase II as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation.

3-Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. Administration of 3-thia fatty acids to rats was followed by significantly increased levels of plasma ketone bodies, whereas the levels of plasma non-esterified fatty acids decreased. The hepatic mRNA levels of fatty acid binding protein and formation of acid-soluble products, using both palmitoyl-CoA and palmitoyl-L-carnitine as substrates, were increased. Hepatic mitochondrial carnitine palmitoyltransferase (CPT) -II and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activities, immunodetectable proteins, and mRNA levels increased in parallel. In contrast, the mitochondrial CPT-I mRNA levels were unchanged and CPT-I enzyme activity was slightly reduced in the liver. The CoA ester of the monocarboxylic 3-thia fatty acid, tetradecylthioacetic acid, which accumulates in the liver after administration, inhibited the CPT-I activity in vitro, but not that of CPT-II. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased, whereas the citrate synthase activity was decreased. The present data suggest that 3-thia fatty acids increase both the transport of fatty acids into the mitochondria and the capacity of the beta-oxidation process. Under these conditions, the regulation of ketogenesis may be shifted to step(s) beyond CPT-I. This opens the possibility that mitochondrial HMG-CoA synthase and CPT-II retain some control of ketone body formation.

Acetates↗

PubMed1999