Biomedical subjects

K Brand

Publications and source records attributed to K Brand.

At least 55 records · Page 3Linked to original sources

PubMed1997

Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death.

Repression of cell cycle progression by tumor suppressors might provide a means for tumor therapy. Here we demonstrate that ectopic overexpression of the p16INK4/CDKN2 tumor suppressor from an adenovirus vector in various cell lines results in block of cell division and, subsequently, in a gradual reduction of the levels of the product of retinoblastoma susceptibility gene, pRb. Overexpression of p53 and p16INK4/CDKN2, but not p53 on its own, induces apoptotic death only in tumor cells. Simultaneous adenoviral transfer of p16 and p53 genes leads to inhibition of tumor growth in nude mice. These results suggest that combined delivery of two cooperating genes like p16 and p53 could be the basis for the development of a new strategy for cancer gene therapy.

Adenoviridae↗

PubMed1997

Role of nuclear factor-kappa B in atherogenesis.

Transcription factors of the nuclear factor-kappa B (NF-kappa B)/Rel family have an important function in the regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. Recent studies strongly indicate that the inducible transcription factor NF-kappa B is involved in the pathogenesis of atherosclerosis. Activated NF-kappa B is present in the fibrotic thickened intima-media and atheromatous areas of the atherosclerotic lesion, within smooth muscle cells, macrophages and endothelial cells, whereas little or no activated NF-kappa B can be detected in vessels lacking atherosclerosis. A variety of molecules have been identified in the atherosclerotic environment that are able to activate NF-kappa B in vitro. Furthermore, an increased expression of numerous genes known to be regulated by NF-kappa B has been found in the atherosclerotic lesion. Possible functional implications for activated NF-kappa B in atherogenesis are discussed here. The activation and role of NF-kappa B in atherosclerosis may provide a model for the involvement of the transcription factor in human chronic inflammatory disease.

Animals↗

PubMed1997

Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein.

Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis.

Antioxidants↗

PubMed1996

The aldolase A promoter in proliferating rat thymocytes is regulated by a cluster of SP1 sites and a distal modulator.

In mitogen-stimulated rat thymocytes the activities and mRNA levels of aldolase A increase remarkably during proliferation pointing to a transcriptional regulation of this enzyme. By DNAse I footprinting and mobility shift competition assays five binding sites for the activating transcription factor Sp1 and one site for an AP-1 like nuclear factor could be identified in the core activating region of the proximal aldolase AH1 promoter downstream of -400. Transfection data and differences found in nuclear protein binding of resting and proliferating cells to DNA sites suggest that Sp1 is an integral part of the mechanism by which the AH1 promoter achieves high level transcription during proliferation. Moreover we demonstrate that an element between positions -1066/-731 significantly attenuates the AH1 promoter driven transcription as well as transcription regulated by the heterologous SV40 promoter. From this effect a functional linkage between the distal muscle-restricted M1 promoter and the active AH1 promoter can be suggested.

Animals↗

PubMed1996

Differences in DNA-binding efficiency of Sp1 to aldolase and pyruvate kinase promoter correlate with altered redox states in resting and proliferating rat thymocytes.

Thymocytes induce their glycolytic enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA-binding efficiency of Sp1 is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Sp1 DNA-binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA-binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12-myristate 13-acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Sp1 transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.

Animals↗

PubMed1996

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Animals↗

PubMed1996

A novel A-->G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency.

We have identified the underlying molecular defect in a patient with hepatic lipase (HL) deficiency presenting with hypertriglyceridemia and premature cardiovascular disease. DNA sequencing of polymerase chain reaction (PCR) amplified DNA and digestion with BsrI established homozygosity for an A-->G mutation in intron I of the patient's hepatic lipase gene. This mutation introduces an additional AG motif within a potential branch lariat signal located 13 bp upstream of the native 3' splice site. Two minigene constructs (normal and mutant) consisting of exons 1 and 2 as well as 192 bp of intron I of HL were generated by the overlap PCR extension method and transfected in human 293 cells. Sequence analysis of reverse transcribed, amplified cDNA generated from total RNA isolated from transfected cells demonstrated the presence of abnormally spliced products containing 13 and 78 additional bases as well as the accumulation of unspliced mRNA. No normally spliced mRNA was identified. Thus, the A-->G mutation disrupts normal splicing of intron I and generates a new AG site that is utilized as an alternative 3' splice signal leading to the most prominent RT-PCR product in vitro. Translation of these alternatively spliced products leads to premature termination resulting in the synthesis of a truncated, non-functional enzyme. The absence of normal HL protein in post heparin plasma of this patient was confirmed by Western blotting. DNA restriction analysis demonstrated that all four of the proband's children, who exhibit HL activity levels between those of the HL-deficient father and the mother with normal HL activity, are heterozygotes for the splice site mutation. Thus, our studies establish the functional significance of a novel mutation in the HL gene of a patient presenting with HL deficiency.

Adenine↗

PubMed1994

Expression of glycolytic isozymes in rat thymocytes during cell cycle progression.

The time courses of activities of aldolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase and pyruvate kinase were determined in stimulated rat thymocytes at 24 h intervals during a period of 72 h of culture. In parallel the mRNA levels of these enzymes were analysed by Northern blotting with specific probes. Both the enzyme activities and the corresponding mRNA levels reached their maxima 48 h after stimulation coinciding with the S-phase of the cell cycle. The isozyme types of aldolase and hexokinase in resting and in mitogen-stimulated rat thymocytes were identified by Northern blot hybridisation using isozyme-specific probes. In these cells the aldolase A is expressed, whereas type B and C could not be detected. The transcription of the aldolase A gene can be regulated by two different promoters. Depending on the alternative usage of the promoters the aldolase A-specific mRNA either contains the non-translated exons M1 or AH1. In rat thymocytes the promoter proximal to the exon AH1 is used while the expression of mRNA I, the type characteristic for muscle tissue, was not observed. In contrast to aldolase two isozyme types of hexokinase were detected. Hexokinase I as well as hexokinase II were present in thymocytes whereas hexokinase III was not detectable. A shift in the isozyme pattern was not observed during the cell cycle progression.

Animals↗

PubMed1994

Glucose is essential for proliferation and the glycolytic enzyme induction that provokes a transition to glycolytic energy production.

A transition from aerobic to anaerobic metabolism occurs as mitogen-activated thymocytes undergo proliferation. Glucose utilization and lactate formation increases 18- and 38-fold, respectively, during proliferation. The absolute amount of 14CO2 production by pyruvate dehydrogenase remains constant, while 14CO2 production by the tricarboxylic acid cycle is reduced during transition from a resting to a proliferating state. Addition of 2,4-dinitrophenol, an agent uncoupling oxidative phosphorylation, and phenacinemethosulfate, an electron acceptor, provide evidence that the reduction of glucose oxidation in proliferating thymocytes is caused neither by limitation of the tricarboxylic acid cycle itself nor by an insufficient supply of ADP. Our data suggest that enhanced cytosolic regeneration of NAD+ by induction of the glycolytic enzymes during proliferation effectively competes with NADH transport and its subsequent oxidation in the mitochondria. Mitogen-stimulated rat thymocytes cultured in a conventional medium containing glucose induce their glycolytic enzymes 8-10-fold in the S phase of the cell cycle and divide within a culture period of 72 h. Replacement of glucose by glutamine, glutamine and ribose, or glutamine and uridine prevents glycolytic enzyme induction and thymocyte proliferation. The effect of glucose on glycolytic enzyme induction cannot be mimicked by 3-O-methylglucose or 2-deoxyglucose. In conclusion, glucose is required for proliferation and the glycolytic enzyme induction that mediates the transition from oxidative to glycolytic energy production during the G1/S transition of rat thymocytes.

3-O-Methylglucose↗

PubMed1994

Inhibition of immediate-early-gene induction in renal mesangial cells by depletion of intracellular polyamines.

Mitogens have been shown to stimulate the activity of the rate-limiting enzyme for polyamine synthesis, ornithine decarboxylase (ODC), and ODC mRNA expression in cultured rat mesangial cells (MCs). In addition, inhibition of ODC by alpha-difluoromethylornithine (DFMO) results in growth arrest of MCs. To elucidate the mechanisms involved in the inhibition of MC proliferation due to polyamine depletion, we studied the effects of DFMO on the activation of phospholipase C and induction of the immediate early genes (IEGs), c-fos, c-jun and Egr-1, which are thought to regulate cell growth. Mitogenic 10% fetal-calf serum (FCS) and 1 unit/ml thrombin activated phospholipase C in MCs within 30 s, as assessed by generation of [3H]inositol phosphates. This activation was not affected by DFMO. mRNAs of the IEGs c-fos, c-jun and Egr-1 were induced by FCS within 15 min. Expression of these genes reached a peak at 60 min and disappeared at 3 h. Treatment of MCs with a growth-suppressing dose of DFMO (5 mM) inhibited mRNAs of all three IEGs by 52-87% at 1 h. Total expression of Egr-1 over 20-120 min was diminished by 41%, and the time point of maximal expression was delayed by 40 min. This inhibitory effect was abolished in a time-dependent manner (1-3 days) by prior addition of 200 microM putrescine, the reaction product of ODC. Egr-1 mRNA expression was super-induced by the inhibitor of protein synthesis, cycloheximide. This effect was also blocked by DFMO. The results indicate that the DFMO-induced process of MC growth inhibition involves steps necessary for IEG activation. The signal-transduction step sensitive to polyamines occurs distal to the activation of phospholipase C. Since reconstitution of normal induction of IEGs requires 3 days, it seems likely that polyamine depletion affects the regulation of IEG expression in an indirect fashion. We conclude that activation of IEGs requires the presence of polyamines and plays a significant role in the induction of MC replication.

Animals↗

PubMed1994

Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity.

T lymphocytes, macrophages, and oxidized low-density lipoprotein (Ox-LDL) are collocalized in early atherosclerotic lesions. Using a low-endotoxin in vitro system, we observed that Ox-LDL but not native LDL induced the production, by both freshly adherent human peripheral blood monocytes and human monocytic THP-1 cells, of the alpha chemokine interleukin (IL)-8, a potent chemoattractant for T lymphocytes. Marked IL-8 induction by Ox-LDL did not require IL-1 beta generation in THP-1 cells. Ox-LDL-induced chemokine production was selective, as Ox-LDL did not stimulate the production by THP-1 cells of the T-lymphocyte chemotactic beta chemokine macrophage inflammatory protein (MIP)-1 alpha. IL-8 induction increased in proportion to the extent of oxidation of LDL as measured by the content of lipid oxidation end products. To identify potentially active components of Ox-LDL, we tested malondialdehyde, an arachidonate-derived lipid oxidation product, and 9-hydroxyoctadecadienoic acid, an oxidation product of linoleate, the major polyunsaturated fatty acid in LDL, and observed that they induced IL-8 generation in the absence of Ox-LDL. Furthermore, when most free lipid oxidation products were removed from Ox-LDL by dialysis, some IL-8-inducing activity was released into the dialysate. However, the major IL-8-inducing activity was not dialyzable. To address the nature of the LDL particle modification required to induce IL-8, acetylated or malondialdehyde-treated native LDL particles were monitored for activity. Neither procedure rendered LDL capable of inducing IL-8. However, phospholipase A2-treated LDL induced THP-1 cell expression of IL-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗

PubMed1994

Oxidized LDL enhances lipopolysaccharide-induced tissue factor expression in human adherent monocytes.

Oxidized low-density lipoprotein (oxLDL) has been characterized as an atherogenic molecule responsible for the induction of a variety of gene products. One such gene, tissue factor (TF), the cellular initiator of the coagulation cascade, is not expressed in normal vascular tissue but is expressed by monocytes and foam cells in atherosclerotic lesions. Therefore, we examined the effect of oxLDL on TF expression in cultured human adherent monocytes. Endotoxin-free oxLDL alone did not induce TF expression in adherent monocytes. However, oxLDL significantly enhanced TF expression induced by the inflammatory mediator, bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In contrast, oxLDL did not alter LPS-mediated production of interleukin-8 and actually inhibited LPS-induced secretion of tumor necrosis factor-alpha, suggesting that some aspects of the signaling pathways for TF induction differ from those of other LPS-responsive monocyte/macrophage gene products. Thus, this study documents specific modulation of the expression of LPS-inducible genes in monocytic cells by oxLDL. Factors that enhance TF expression in monocyte/macrophage cells present in atheroma may contribute to the severity of thrombotic episodes and complications observed in atherosclerosis.

Cells, Cultured↗

PubMed1993

Mass changes of inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes.

Changes in the cellular mass and the cellular concentration of the highly phosphorylated inositol derivatives inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) and inositol hexakisphosphate (InsP6) were observed during a complete cell cycle of proliferating rat thymocytes. Inositol polyphosphates were determined by a recently developed anion-exchange HPLC/post-column complexometric dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591), and data were expressed as mass amounts per cell number or as absolute intracellular concentrations based on cell volume determinations by the cell analyzer system CASY 1. After a very early transient rise in both Ins(1,3,4,5,6)P5 and InsP6 at the beginning of the cell cycle, a decrease of the intracellular concentration of both compounds occurred which then remained on a low level between 24 and 48 h. Between 48 and 72 h the cells divided resulting in a decrease of the cell volume by a factor of approximately 2. During this phase a pronounced increase of Ins(1,3,4,5,6)P5 and InsP6 was observed. These increases were already seen when the data were expressed as mass per cell number, but were even enhanced when expressed as absolute intracellular concentrations. In conclusion, we present as a novel finding long term changes in the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 in proliferating thymocytes. This may indicate a role for these compounds during cell cycle progression.

Animals↗

PubMed1993

Cell cycle-related expression of poly(ADP-ribosyl)transferase in proliferating rat thymocytes.

The activity profile of poly(ADP-ribosyl)transferase was assayed during a complete cell cycle of rat thymocytes stimulated in the presence of interleukin-2 by concanavalin A or monoclonal antibodies against the T-cell antigen receptor (TCRmAB). Poly ADP-ribosylation was measured in permeabilized cells by the incorporation of [adenine-3H]NAD+ into protein bound poly ADP-ribose. The polymers of ADP-ribose were separated from the monomers using dihydroxyboronyl-Bio-Rex 70 columns. The rate of poly(ADP-ribosyl)ation increases during the G1 phase with a maximum 12 h after stimulation. This increase in activity is due to enhanced de novo synthesis of poly(ADP-ribosyl)transferase which can be abolished by the addition of cycloheximide. The half-life of this enzyme during the induction period was estimated to be 4 h. A second activity peak appears during the S-phase of the cell cycle 48 h after stimulation. The maxima of the poly(ADP-ribosyl)ation rate coincide with elevated immunoreactive enzyme levels at 12 and 48 h of culture assayed by Western blotting. The mRNA levels of pADPRT do not correlate with the first maximum of activity, whereas the second maximum was accompanied by a 5-fold increase of the specific mRNA. These results suggest a translational regulation of pADPRT in the G1 phase of the cell cycle, whereas the second activity peak in the S-phase is due to an increased transcription and translation. The induction of pADPRT activity in the G1 phase of TCRmAB-stimulated cells points to a function of poly(ADP-ribosyl)ation in the proliferation of thymocytes.

Adenosine Diphosphate Ribose↗

PubMed1993

The role of the Crabtree effect and an endogenous fuel in the energy metabolism of resting and proliferating thymocytes.

Rat thymocytes have been used to characterize the changes in energy metabolism that occur as cells undergo a resting/proliferation transition. In the resting state, anaerobic ATP production accounts for only 4% of ATP turnover. The remainder is fueled by the oxidation of a mixture of an unidentified endogenous fuel (62%), glucose (18%) and glutamine (16%). 48 h after mitogen stimulation, the ATP turnover has increased twofold. In these proliferating cells, glucose inhibits oxygen consumption by 58%, indicating a profound Crabtree effect which is not present in resting cells. Consequently, proliferating cells, in the presence of glucose and glutamine, fuel the majority (61%) of ATP turnover anaerobically, producing lactate from glucose. The development of a Crabtree effect may be the result of the 8-10-fold increase in glycolytic enzyme activities which occurs with proliferation. Possible advantages of such a proliferative metabolism are a sparing of endogenous fuel, and a minimizing of oxidative metabolism, with its concurrent production of free radicals.

Adenosine Triphosphate↗

PubMed1993

Interferon-gamma inhibits macrophage apolipoprotein E production by posttranslational mechanisms.

Macrophage-derived apolipoprotein (apo) E and multimers of a synthetic apo E-peptide display monokine-like functions by inhibiting mitogen- or antigen-driven lymphocyte proliferation. This study demonstrated how the target lymphocyte itself can modulate macrophage apo E production. The lymphokine interferon-gamma (IFN) dramatically inhibited the accumulation of apo E in the supernatant of human monocytic THP-1 cells when present during phorbol myristate acetate-induced differentiation. A similar effect was observed when IFN was added to differentiated THP-1 cells. Treatment with IFN did not change the steady-state levels of apo E mRNA. Furthermore, in the presence of IFN no increased degradation or increased uptake of extracellular apo E was detected. Pulse-chase experiments indicated that IFN reduced the accumulation of extracellular apo E and increased the degradation of intracellular apo E. The inhibitory effect of IFN on apo E production also was observed in human monocyte-derived macrophages. Thus, our data demonstrated that IFN inhibited macrophage apo E production by posttranslational mechanisms. This represents a previously uncharacterized immunoregulatory interaction and lends further support to a relationship between lipid metabolism and the immune system.

Apolipoproteins E↗

PubMed1992

Cell cycle-associated expression of M2-type isozyme of pyruvate kinase in proliferating rat thymocytes.

During a complete cell cycle of rat thymocytes stimulated by concanavalin A and interleukin 2, the activity and mRNA level of pyruvate kinase reached a maximum (8-12-fold increase) 48 h after stimulation coinciding with the S-phase of the cell cycle. Increases of cellular enzyme activity, pyruvate kinase protein, and mRNA levels are correlated up to 48 h of culture. Afterwards pyruvate kinase activity and mRNA levels decrease, whereas the pyruvate kinase protein continues to increase throughout mitosis. This change of specific pyruvate kinase activity points to a posttranslational modification of the enzyme besides its transcriptional regulation. The presence of the M2-type isozyme was determined by the following methods: (a) native cellulose acetate electrophoresis and activity staining, (b) Northern blot hybridization with M1- and M2-specific cDNA probes, and (c) determination of kinetic parameters. The isozyme pattern did not change during the cell cycle progression. The induction of pyruvate kinase is completely abolished by 2-difluoromethylornithine-mediated polyamine depletion. However, the proportion of hybridizable pyruvate kinase mRNA was not affected. These data suggest the requirement of polyamines for efficient translation rather than transcription during cell growth.

Animals↗

PubMed1992

Control of cell-cycle-associated tetrahydrobiopterin synthesis in rat thymocytes.

The cell-cycle progression of rat thymocytes from G0 through G1 to DNA synthesis is associated with a transient synthesis of H4biopterin, the concentration of which reaches a maximum at the time of S-phase entry and then decreases. This synthesis of H4biopterin is controlled by the specific activity of GTP cyclohydrolase I, which peaks in G1/S cells. In contrast, the catalytic activity of sepiapterin reductase remains constant throughout the cell-cycle. At G0 the steady state mRNA levels specific for GTP cyclohydrolase I and sepiapterin reductase, respectively, are below the limits of detection. Both accumulate as the thymocytes progress through the cell-cycle but lack cyclic down regulation. The data indicate that the variations in H4biopterin synthesis during the cell-cycle are caused by growth regulated increase in GTP cyclohydrolase I mRNA expression, with subsequent post-translational inactivation. This latter is likely due to the degree of enzyme phosphorylation.

Alcohol Oxidoreductases↗