Papers by Frank Oerlemans
Advances in experimental medicine and biology, 1984
The frequency of MAD deficiency in cases with exercise intolerance compared with the frequency in... more The frequency of MAD deficiency in cases with exercise intolerance compared with the frequency in series of consecutive muscle biopsies suggests a relation between the deficiency and exercise intolerance. Deficiency cases can be presumed by an impaired NH3 production during ischaemic exercise. The ischaemic exercise test also gives information concerning the familial character of the deficiency.
Clinical Science, 1986
1. Plasma adenosine, inosine and hypoxanthine concentrations were assayed in seven control subjec... more 1. Plasma adenosine, inosine and hypoxanthine concentrations were assayed in seven control subjects, five myoadenylate deaminase deficient (MADD) patients and six McArdle patients before and after ischaemic forearm exercise. 2. The plasma adenosine increase was very low in all test groups and there were no significant differences. 3. The MADD patients showed a significantly lower increase of plasma inosine and hypoxanthine after exercise as compared with the controls. 4. In the McArdle patients the increase in plasma inosine and hypoxanthine after exercise did not differ significantly from the values measured in the controls. 5. The ischaemic exercise test with measurement of plasma inosine and hypoxanthine might be of diagnostic value in MADD, but not in McArdle's disease.
Biochemical and Biological …, 1980
Advances in Experimental Medicine and Biology, 1980
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Acta Neurobiologiae Experimentalis, 2003
Cell
Male patients with fragile X syndrome lack FMRl pro-tein due to silencing of the FMRl gene by amp... more Male patients with fragile X syndrome lack FMRl pro-tein due to silencing of the FMRl gene by amplification of a CGG repeat and subsequent methylation of the promoter region. The absence of FMRl protein leads to mental retardation, aberrant behavior, and macroor-chidism. Hardly anything is known about the physiologi-cal function of FMR1 and the pathological mechanisms leading to these symptoms. Therefore, we designed a knockout model for the fragile X syndrome in mice. The knockout mice lack normal Fmrl protein and show mac-roorchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR7 and the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.
Journal of Clinical & Cellular Immunology, 2015
Background: Macrophages are highly specialized immune cells of different developmental origin, wh... more Background: Macrophages are highly specialized immune cells of different developmental origin, which occur in a continuum of diverse functional states in almost all tissues. In order to fulfil their complex role in tissue homeostasis and defence against pathogens they must be able to live with heterogeneous extrinsic nutrient conditions in tissue niches and handle variation in intrinsic metabolic demand that is determined by differentiation state, functional specialization and immune challenge. The purpose of the present study was to gain more insight in how metabolic specialization and versatility in fate and immune effector function of macrophages are coupled. Methods: In vitro phenotypic characteristics of two macrophage cell lineages of profoundly different developmental origin and polarization capacity, RAW 264.7 and Maf-DKO cells, were analysed. By use of biochemical and cell biological approaches and scanning electron microscopy, we studied the metabolic profiles of these two types of macrophages in relation to proliferative capacity, morphological appearance of cell surface and cell shape, and phagocytic activity as index of morphodynamic potential. Results: Comparison of gross features of carbohydrate metabolism, including levels of glycolytic enzymes Hexokinase (HK), Pyruvate Kinase (PK-M2), lactate dehydrogenase and nicotinamide phosphoribosyltransferase (Nampt), glucose and oxygen consumption and lactate production rates, and intracellular concentrations and redox ratios of NAD(P)(H) demonstrated that RAW 264.7 and Maf-DKO cells are conspicuously similar in that they both rely heavily on the use of glycolysis. In this respect they share many characteristics with primary macrophages. Strikingly, this uniform metabolic signature does not translate in behavioral-functional similarities as RAW 264.7 cells have a significantly higher proliferation rate, whereas Maf-DKO cells appear to be morphodynamically more active, form significantly more surface membrane protrusions and phagocytose complement opsonized particles much more efficiently. Conclusion: We conclude that the global rate of glycolysis in intermediary carbohydrate metabolism is similar for the two cell lineages, but that they can make differential use of this important pathway, either to fuel high morphodynamic activity in Maf-DKO cells, or for the sustenance of cell growth in fast proliferating RAW 264.7 cells. Our findings are in keeping with the idea that macrophages may uniformly prefer use of the rapid response time of glycolysis because this pathway provides them with the ability to meet any possible short-timescale energy demand required for immune function.
Physiology & Behavior, 2009
The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and U... more The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0°C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK−−/− − mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK−−/−− mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect.
Signalling Mechanisms — from Transcription Factors to Oxidative Stress, 1995
Receptor-like protein tyrosine phosphatases (RPTPases) comprise an extracellular ligand-binding r... more Receptor-like protein tyrosine phosphatases (RPTPases) comprise an extracellular ligand-binding region, a transmembrane domain, and as a rule two cytoplasmic tyrosine phosphatase domains. In vitro studies using the cytoplasmic parts of RPTPases and artificial substrates have suggested that the first, membrane proximal phosphatase domain exhibits catalytic activity, whereas the second, C-terminal phosphatase domain may regulate the phosphatase activity of the first domain. Further studies, however, are hampered by the fact that RPTPase-specific ligands and substrates still remain to be identified. Also, the complexity of transmembrane signalling is difficult to mimick in vitro. To circumvent these problems, the individual functions of the two phosphatase domains in RPTPases can be studied in vivo by means of homologous recombination in mouse embryonic stem (ES) cells. Here, we decribe the use of ’double replacement’ gene targeting in mouse embryonic stem cells to generate cell and animal models for studying the individual role of both phosphatase domains of the RPTPase Leukocyte common Antigen-Related molecule LAR. In addition, exploiting the process of gene conversion, LAR-negative ES cells were generated to enable structure-function analysis of LAR mutants on a null background.
PLoS ONE, 2014
Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu... more Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics.
PLoS ONE, 2009
Background: Creatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate group... more Background: Creatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. Methodology/Principal Findings: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. Conclusion/Significance: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to onsite availability of ATP generated by CK-B.
PLoS ONE, 2014
Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin c... more Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD + and NADH) and NADP(H) (i.e. NADP + and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD +supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD + in macrophages. Inhibition of NAD + salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD + levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD + induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD + levels by NAD + , NMN, or NADP + supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD + 's cytosolic role in the regulation of morphofunctional characteristics of macrophages.
PLoS Biology, 2008
Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerizat... more Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and ßactin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-yR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced Factin assembly events in early phagocytosis by providing local ATP supply.
Physiological Genomics, 2004
Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological f... more Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the regulation of microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. To investigate the biological function of PTP-BL enzyme activity, we have generated mice that lack the PTP-BL PTP moiety. These PTP-BLΔP/ΔPmice are viable and fertile and do not present overt morphological alterations. Although PTP-BL is expressed in most hematopoietic cell lineages, no alterations of thymocyte development in PTP-BLΔP/ΔPmice could be detected. Sciatic nerve lesioning revealed that sensory nerve recovery is unaltered in these mice. In contrast, a very mild but significant impairment of motor nerve repair was observed. Our findings exclude an essential role for PTP-BL as a phosphotyrosine phosphatas...
Nucleic Acids Research, 1991
The cytosolic creatine klnases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have... more The cytosolic creatine klnases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue-and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunlts, starting at the muscle creatine kinase (CKM) gene which is transcriptlonally Inactive during early embryogenesls. Using repeated rounds of gene targeting In mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycln resistance (neo^ gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an Insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several Independent doubly targeted clones were isolated which all had insertions in the previously neo-dlsrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted In gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels In ES cells, led to the formation of the BM isoform of CK in these clones.
Nature Medicine, 2008
Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequentl... more Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-jB ligand in cooperation with macrophage colony-stimulating factor 1-3. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/mice were similarly affected. In vivo studies showed that Ckb-/mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.
Molecular Cancer, 2009
Background The Warburg phenotype in cancer cells has been long recognized, but there is still lim... more Background The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. Results Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux incr...
Molecular and Cellular Biochemistry, 2004
Brain-type creatine kinases B-CK (cytosolic) and UbCKmit (mitochondrial) are considered important... more Brain-type creatine kinases B-CK (cytosolic) and UbCKmit (mitochondrial) are considered important for the maintenance and distribution of cellular energy in the central nervous system. Previously, we have demonstrated an abnormal behavioral phenotype in mice lacking the B-CK creatine kinase isoform, regarding exploration, habituation, seizure susceptibility and spatial learning. The phenotype in these mice was associated with histological adaptations in the hippocampal mossy fiber field size. Here, mice lacking the ubiquitous mitochondrial creatine kinase isoform (UbCKmit-/-mice) showed, when subjected to a similar battery of behavioral tasks, diminished open field habituation and slower spatial learning acquisition in the Morris water maze task, but normal sensory or motor functions. A reduced acoustic startle response, higher threshold, and lack of prepulse inhibition were observed in UbCKmit-/-mice, suggesting that the unconditioned reflexive responsiveness is not optimal. Our findings suggest a role for mitochondrial CK-mediated high-energy phosphoryl transfer in synaptic signalling in the acoustic signal response network and hippocampal-dependent learning circuitry of brain. Finally, we demonstrate that UbCKmit has a widespread occurrence in the cell soma of neuronal nuclei along the rostro-caudal axis of the brain, i.e. cortex, midbrain, hindbrain, cerebellum and brainstem, similar to the occurrence of B-CK. This may explain the similarity of phenotypes in mice lacking B-CK or UbCKmit. We predict that the remaining functional intactness of the cytosolic B-CK reaction and perhaps the compensatory role of other phosphoryl transfer systems are sufficient to sustain the energy requirements for basic sensory, motor and physiological activities in UbCKmit-/-mice.
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Papers by Frank Oerlemans